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• W1976779503 abstract "Background & Aims: There is evidence that, in Crohn’s disease (CD), lamina propria T lymphocytes (LPLs) are resistant to FAS-mediated apoptosis and that this defect contributes to the mucosal T-cell accumulation. In this study we examined the functional role of Flip, a Flice inhibitor protein, in the resistance of CD LPL to FAS-mediated apoptosis. Methods: Biopsy specimens and LPLs were taken from CD and ulcerative colitis (UC) patients and normal controls and analyzed for Flip by Western blotting. We also examined whether inhibition of Flip by antisense oligonucleotide restored the susceptibility of CD LPLs to FAS-induced apoptosis. LPL apoptosis was assessed by flow cytometry. Results: After FAS stimulation, the rate of apoptosis of CD3+ LPLs was higher in normal controls and patients with UC than in patients with CD. Enhanced expression of both long and short Flip isoforms was seen in biopsy specimens and purified CD3+ and CD45RO+ LPLs of CD patients in comparison with UC patients and normal controls. No increase in Flip was documented in untreated celiac disease mucosa, thus suggesting the possibility that induction of Flip in the gut does not simply rely on the ongoing inflammation. Finally, we showed that inhibition of Flip by antisense oligonucleotide reverted the resistance of CD LPLs to FAS-induced apoptosis. Conclusions: Data suggest a role for Flip in the resistance of CD LPLs to FAS-mediated apoptosis. Background & Aims: There is evidence that, in Crohn’s disease (CD), lamina propria T lymphocytes (LPLs) are resistant to FAS-mediated apoptosis and that this defect contributes to the mucosal T-cell accumulation. In this study we examined the functional role of Flip, a Flice inhibitor protein, in the resistance of CD LPL to FAS-mediated apoptosis. Methods: Biopsy specimens and LPLs were taken from CD and ulcerative colitis (UC) patients and normal controls and analyzed for Flip by Western blotting. We also examined whether inhibition of Flip by antisense oligonucleotide restored the susceptibility of CD LPLs to FAS-induced apoptosis. LPL apoptosis was assessed by flow cytometry. Results: After FAS stimulation, the rate of apoptosis of CD3+ LPLs was higher in normal controls and patients with UC than in patients with CD. Enhanced expression of both long and short Flip isoforms was seen in biopsy specimens and purified CD3+ and CD45RO+ LPLs of CD patients in comparison with UC patients and normal controls. No increase in Flip was documented in untreated celiac disease mucosa, thus suggesting the possibility that induction of Flip in the gut does not simply rely on the ongoing inflammation. Finally, we showed that inhibition of Flip by antisense oligonucleotide reverted the resistance of CD LPLs to FAS-induced apoptosis. Conclusions: Data suggest a role for Flip in the resistance of CD LPLs to FAS-mediated apoptosis. Induction of apoptosis or programmed cell death is one of the mechanisms by which the immune system negatively regulates the expansion of activated cell clones.1Marrack P. Kappler J. Control of T cell viability.Annu Rev Immunol. 2004; 22: 765-787Crossref PubMed Scopus (314) Google Scholar, 2Rathmell J.C. Thompson C.B. Pathways of apoptosis in lymphocyte development, homeostasis, and disease.Cell. 2002; 109: S97-S107Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 3Green D.R. Overview apoptotic signaling pathways in the immune system.Immunol Rev. 2003; 193: 5-9Crossref PubMed Scopus (101) Google Scholar This process appears to be particularly relevant at the intestinal level where T lymphocytes continuously are under the stimulus of luminal antigens.4Bu P. Keshavarzian A. Stone D.D. Liu J. Le P.T. Fisher S. Qiao L. Apoptosis one of the mechanisms that maintains unresponsiveness of the intestinal mucosal immune system.J Immunol. 2001; 166: 6399-6403Crossref PubMed Scopus (67) Google Scholar Indeed, studies from several laboratories have shown previously that normal lamina propria T lymphocytes (LPLs) spontaneously undergo apoptosis and that challenge of LPLs with activating stimuli results in enhanced cell apoptosis.5Boirivant M. Pica R. DeMaria R. Testi R. Pallone F. Strober W. Stimulated human lamina propria T cells manifest enhanced Fas-mediated apoptosis.J Clin Invest. 1996; 98: 2616-2622Crossref PubMed Scopus (123) Google Scholar, 6De Maria R. Boirivant M. Cifone M.G. Roncaioli P. Hahne M. Tschopp J. Pallone F. Santoni A. Testi R. Functional expression of Fas and Fas ligand on human gut lamina propria T lymphocytes. A potential role for the acidic sphingomyelinase pathway in normal immunoregulation.J Clin Invest. 1996; 97: 316-322Crossref PubMed Scopus (119) Google Scholar, 7Sturm A. Mohr S. Fiocchi C. Critical role of caspases in the regulation of apoptosis and proliferation of mucosal T cells.Gastroenterology. 2002; 122: 1334-1345Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar T-cell apoptosis is in part dependent on the activity of an intracellular pathway that is triggered by the interaction of FAS ligand with FAS, a 50-kilodalton cysteine-rich transmembrane protein belonging to the tumor necrosis factor–receptor family.8Hofmann K. The modular nature of apoptotic signaling proteins.Cell Mol Life Sci. 1999; 55: 1113-1128Crossref PubMed Scopus (125) Google Scholar, 9Locksley R.M. Killeen N. Lenardo M.J. The TNF and TNF receptor superfamilies integrating mammalian biology.Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (3018) Google Scholar, 10Thome M. Tschopp J. Regulation of lymphocyte proliferation and death by FLIP.Nat Rev Immunol. 2001; 1: 50-58Crossref PubMed Scopus (352) Google Scholar, 11Alderson M.R. Tough T.W. Davis-Smith T. Braddy S. Falk B. Schooley K.A. Goodwin R.G. Smith C.A. Ramsdell F. Lynch D.H. Fas ligand mediates activation-induced cell death in human T lymphocytes.J Exp Med. 1995; 181: 71-77Crossref PubMed Scopus (868) Google Scholar The intracellular portion of FAS has a region of about 80 residues that is required for death signaling and has been called the death domain. The death domain interacts with an adaptor protein called the FAS-associated death domain, whose N-terminal domain, called the death effector domain (DED), is necessary for the induction of death. The DED of the FAS-associated death domain recruits 2 DED-containing caspases, caspase-8 and caspase-10, to form the death-inducing signaling complex, thereby initiating apoptosis.9Locksley R.M. Killeen N. Lenardo M.J. The TNF and TNF receptor superfamilies integrating mammalian biology.Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (3018) Google Scholar After its activation, caspase 8 can trigger 2 distinct cell death pathways by either promoting mitochondrial cytochrome c release or bypassing mitochondria and activating the executioner caspases directly, such as caspase 3.12Peter M.E. Krammer P.H. The CD95(APO-1/Fas) DISC and beyond.Cell Death Differ. 2003; 10: 26-35Crossref PubMed Scopus (894) Google Scholar, 13Green D.R. Apoptotic pathways paper wraps stone blunts scissors.Cell. 2000; 102: 1-4Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar The role of the FAS signaling pathway in controlling abnormal expansion of autoreactive T cells is supported by the demonstration that defects in FAS-mediated cell apoptosis are seen in several immune-mediated diseases characterized by an accumulation of T cells within the inflamed tissue. For example, a diminished FAS-mediated cell death occurs in patients with multiple sclerosis and autoimmune thyroiditis.14Comi C. Leone M. Bonissoni S. DeFranco S. Bottarel F. Mezzatesta C. Chiocchetti A. Perla F. Monaco F. Dianzani U. Defective T cell fas function in patients with multiple sclerosis.Neurology. 2000; 55: 921-927Crossref PubMed Scopus (105) Google Scholar, 15Eguchi K. Apoptosis in autoimmune diseases.Intern Med. 2001; 40: 275-284Crossref PubMed Scopus (136) Google Scholar, 16Stassi G. Di Liberto D. Todaro M. Zeuner A. Ricci-Vitiani L. Stoppacciaro A. Ruco L. Farina F. Zummo G. De Maria R. Control of target cell survival in thyroid autoimmunity by T helper cytokines via regulation of apoptotic proteins.Nat Immunol. 2000; 1: 483-488Crossref PubMed Scopus (133) Google Scholar Similarly, a defective FAS-mediated apoptosis has been documented in patients with Crohn’s disease (CD), a chronic inflammatory disorder of the gastrointestinal tract, characterized by a tissue damaging T-cell–dependent immune response.17Ina K. Itoh J. Fukushima K. Kusugami K. Yamaguchi T. Kyokane K. Imada A. Binion D.G. Musso A. West G.A. Dobrea G.M. McCormick T.S. Lapetina E.G. Levine A.D. Ottaway C.A. Fiocchi C. Resistance of Crohn’s disease T cells to multiple apoptotic signals is associated with a Bcl-2/Bax mucosal imbalance.J Immunol. 1999; 163: 1081-1090PubMed Google Scholar, 18Fiocchi C. Inflammatory bowel disease etiology and pathogenesis.Gastroenterology. 1998; 115: 182-205Abstract Full Text Full Text PDF PubMed Scopus (1865) Google Scholar, 19Boirivant M. Marini M. Di Felice G. Pronio A.M. Montesani C. Tersigni R. Strober W. Lamina propria T cells in Crohn’s disease and other gastrointestinal inflammation show defective CD2 pathway-induced apoptosis.Gastroenterology. 1999; 116: 557-565Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar Importantly, in CD tissue, LPLs express FAS and mucosal cells contain normal levels of FAS ligand, thus suggesting that failure of CD LPLs to respond to FAS-induced apoptosis may be secondary to changes in the expression and/or activity of intracellular molecules that participate in the FAS-mediated cell death program.20Ueyama H. Kiyohara T. Sawada N. Isozaki K. Kitamura S. Kondo S. Miyagawa J. Kanayama S. Shinomura Y. Ishikawa H. Ohtani T. Nezu R. Nagata S. Matsuzawa Y. High Fas ligand expression on lymphocytes in lesions of ulcerative colitis.Gut. 1998; 43: 48-55Crossref PubMed Scopus (100) Google Scholar, 21Melgar S. Hammarstrom S. Oberg A. Danielsson A. Hammarstrom M.L. Cytolytic capabilities of lamina propria and intraepithelial lymphocytes in normal and chronically inflamed human intestine.Scand J Immunol. 2004; 60: 167-177Crossref PubMed Scopus (17) Google Scholar In this context, it has been shown recently that bcl-2, an anti-apoptotic protein, is up-regulated in CD LPLs.17Ina K. Itoh J. Fukushima K. Kusugami K. Yamaguchi T. Kyokane K. Imada A. Binion D.G. Musso A. West G.A. Dobrea G.M. McCormick T.S. Lapetina E.G. Levine A.D. Ottaway C.A. Fiocchi C. Resistance of Crohn’s disease T cells to multiple apoptotic signals is associated with a Bcl-2/Bax mucosal imbalance.J Immunol. 1999; 163: 1081-1090PubMed Google Scholar, 19Boirivant M. Marini M. Di Felice G. Pronio A.M. Montesani C. Tersigni R. Strober W. Lamina propria T cells in Crohn’s disease and other gastrointestinal inflammation show defective CD2 pathway-induced apoptosis.Gastroenterology. 1999; 116: 557-565Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar Although, the high bcl-2 levels might protect mitochondria and prevent cell death, this would be insufficient to explain the resistance of CD LPLs to FAS-mediated apoptosis. In fact, it is well known that in T cells, FAS-activated caspase-8 directly can cleave executioner caspases and cause death when mitochondria are protected by bcl-2.22Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. Two CD95 (APO-1/Fas) signaling pathways.EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2631) Google Scholar, 23Muppidi J.R. Tschopp J. Siegel R.M. Life and death decisions secondary complexes and lipid rafts in TNF receptor family signal transduction.Immunity. 2004; 21: 461-465Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar The FAS-associated-death domain–like interleukin-1β–converting enzyme (Flice)-inhibitory protein (Flip) is an intracellular inhibitor of the FAS-induced cell death cascade.10Thome M. Tschopp J. Regulation of lymphocyte proliferation and death by FLIP.Nat Rev Immunol. 2001; 1: 50-58Crossref PubMed Scopus (352) Google Scholar, 24Irmler M. Thome M. Hahne M. Schneider P. Hofmann K. Steiner V. Bodmer J.L. Schroter M. Burns K. Mattmann C. Rimoldi D. French L.E. Tschopp J. Inhibition of death receptor signals by cellular FLIP.Nature. 1997; 388: 190-195Crossref PubMed Scopus (2227) Google Scholar Because of an alternative splicing, 2 gene products can be translated in 2 different isoforms: a larger isoform, called Flip Long (FlipL), which possesses 2 DED and a caspase-like domain, and a smaller protein, called Flip Short (FlipS), that possesses 2 DED, but no caspase-like domain.10Thome M. Tschopp J. Regulation of lymphocyte proliferation and death by FLIP.Nat Rev Immunol. 2001; 1: 50-58Crossref PubMed Scopus (352) Google Scholar On FAS-activating stimuli, Flip is recruited at the death-inducing signaling complex level where it prevents the cleavage of procaspase-8, thereby inhibiting the activation of caspase-8.25Scaffidi C. Schmitz I. Krammer P.H. Peter M.E. The role of c-FLIP in modulation of CD95-induced apoptosis.J Biol Chem. 1999; 274: 1541-1548Crossref PubMed Scopus (711) Google Scholar, 26Krueger A. Schmitz I. Baumann S. Krammer P.H. Kirchhoff S. Cellular FLICE-inhibitory protein splice variants inhibit different steps of caspase-8 activation at the CD95 death-inducing signaling complex.J Biol Chem. 2001; 276: 20633-20640Crossref PubMed Scopus (483) Google Scholar Consistent with this, enhanced expression of Flip has been documented in chronic inflammatory diseases characterized by a defective FAS-induced apoptosis.14Comi C. Leone M. Bonissoni S. DeFranco S. Bottarel F. Mezzatesta C. Chiocchetti A. Perla F. Monaco F. Dianzani U. Defective T cell fas function in patients with multiple sclerosis.Neurology. 2000; 55: 921-927Crossref PubMed Scopus (105) Google Scholar, 15Eguchi K. Apoptosis in autoimmune diseases.Intern Med. 2001; 40: 275-284Crossref PubMed Scopus (136) Google Scholar, 16Stassi G. Di Liberto D. Todaro M. Zeuner A. Ricci-Vitiani L. Stoppacciaro A. Ruco L. Farina F. Zummo G. De Maria R. Control of target cell survival in thyroid autoimmunity by T helper cytokines via regulation of apoptotic proteins.Nat Immunol. 2000; 1: 483-488Crossref PubMed Scopus (133) Google Scholar, 27Semra Y.K. Seidi O.A. Sharief M.K. Overexpression of the apoptosis inhibitor FLIP in T cells correlates with disease activity in multiple sclerosis.J Neuroimmunol. 2001; 113: 268-274Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 28Sharief M.K. Increased cellular expression of the caspase inhibitor FLIP in intrathecal lymphocytes from patients with multiple sclerosis.J Neuroimmunol. 2000; 111: 203-209Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 29Djerbi M. Abdul-Majid K.B. Abedi-Valugerdi M. Olsson T. Harris R.A. Grandien A. Expression of the long form of human FLIP by retroviral gene transfer of hemopoietic stem cells exacerbates experimental autoimmune encephalomyelitis.J Immunol. 2003; 170: 2064-2073Crossref PubMed Scopus (18) Google Scholar, 30Perlman H. Pagliari L.J. Georganas C. Mano T. Walsh K. Pope R.M. FLICE-inhibitory protein expression during macrophage differentiation confers resistance to fas-mediated apoptosis.J Exp Med. 1999; 190: 1679-1688Crossref PubMed Scopus (219) Google Scholar, 31Perlman H. Pagliari L.J. Liu H. Koch A.E. Haines 3rd, G.K. Pope R.M. Rheumatoid arthritis synovial macrophages express the Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein and are refractory to Fas-mediated apoptosis.Arthritis Rheum. 2001; 44: 21-30Crossref PubMed Scopus (102) Google Scholar, 32Schedel J. Gay R.E. Kuenzler P. Seemayer C. Simmen B. Michel B.A. Gay S. FLICE-inhibitory protein expression in synovial fibroblasts and at sites of cartilage and bone erosion in rheumatoid arthritis.Arthritis Rheum. 2002; 46: 1512-1518Crossref PubMed Scopus (80) Google Scholar Based on these observations, we hypothesized that high Flip levels could contribute to the resistance of CD LPLs to FAS-mediated apoptosis. In the present study, we therefore analyzed the expression and role of Flip in CD. Biopsy specimens were obtained from the colons of 15 patients with moderate to severe CD undergoing endoscopy. In all of these patients, the primary site of involvement of the disease was the terminal ileum and colon. Eight patients were receiving corticosteroids, and the remaining patients were receiving mesalazine. Mucosal samples also were taken from intestinal resection specimens of 13 patients with moderate to severe CD. One patient was taking antibiotics and the other patients were on corticosteroids. In all patients, the indication for surgery was a chronic active course of disease that was poorly responsive to medical treatment. Paired biopsy specimens were taken from inflamed and uninflamed mucosal areas of 6 of 11 CD patients undergoing endoscopy. Mucosal samples also were taken from 10 patients with active ulcerative colitis (UC) undergoing endoscopy. Six of these patients were receiving corticosteroids, and the remaining patients were on mesalazine. Mucosal samples also were available from 6 UC patients undergoing colectomy for a chronic active course of disease that was poorly responsive to medical treatment. All UC patients were receiving corticosteroids. Controls included normal colonic mucosal biopsy specimens from 14 patients with irritable bowel syndrome, and macroscopically and microscopically unaffected colonic specimens from 16 patients with colon cancer. In addition, duodenal biopsy specimens were taken from 6 patients with untreated celiac disease and 6 normal controls. The histopathologic diagnosis of celiac disease was based on typical mucosal lesions with crypt cell hyperplasia, villous atrophy, and an increased number of intraepithelial leukocytes. All untreated celiac disease patients were positive for anti-endomysial and antitransglutaminase antibodies at the time of tissue collection. No patient with refractory celiac disease was enrolled in this study. Age-matched normal controls were under investigation for gastrointestinal symptoms, but had normal histology, no increase in inflammatory cells, and were anti-endomysial–antibody and antitransglutaminase-antibody negative. Lamina propria mononuclear cells (LPMCs) were prepared as described previously.33Monteleone G. Biancone L. Marasco R. Morrone G. Marasco O. Luzza F. Pallone F. Interleukin 12 is expressed and actively released by Crohn’s disease intestinal lamina propria mononuclear cells.Gastroenterology. 1997; 112: 1169-1178Abstract Full Text PDF PubMed Scopus (499) Google Scholar An aliquot of LPMCs was used immediately for the analysis of Flip. The remaining cells were used to purify CD3+ LPLs or resuspended in RPMI 1640 (Sigma, Milan, Italy) and supplemented with 10% fetal calf serum (Sigma). CD3+ and CD45RO+ LPLs were isolated from total LPMCs by MACS separators (Miltenyi Biotec, Caldera di Reno, Italy) using CD3+ and CD45RO+ T-cell isolation kits. To examine the response of normal, UC, and CD LPLs to apoptotic stimuli, LPMCs either were left unstimulated or stimulated with a human monoclonal anti–FAS-activating antibody (clone CH11; Upstate Biotechnology Inc, Lake Placid, NY; final concentration, 200 ng/mL) or anti-CD3 (clone OKT3, 2% final dilution)34Monteleone G. MacDonald T.T. Wathen N.C. Pallone F. Pender S.L. Enhancing lamina propria Th1 cell responses with interleukin 12 produces severe tissue injury.Gastroenterology. 1999; 117: 1069-1077Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar and anti-CD2 antibody for 24 hours. The anti-CD2 monoclonal antibody pair (T112 and T113; Dana-Farber Cancer Institute, Boston, MA) was used in soluble form at a 1:1000 final dilution. At the end of culture, cells were harvested and the percentage of Annexin-V/CD3+ was assessed by flow cytometry. To evaluate if Flip is regulated by in vitro activation of LPLs, normal, UC, and CD LPMCs were cultured in the presence or absence of anti-CD3 or anti-CD3 and anti-CD2 for 24 hours. At the end, cell extracts were prepared and analyzed for the content of Flip and interleukin (IL)-21 by Western blotting. To evaluate the role of Flip in the CD LPL resistance to FAS-mediated apoptosis, CD, UC, and normal LPMCs were cultured (1 × 106/mL) in the presence or absence of a specific Flip antisense (GACTTCAGCAGACATCCTAC-3′) or control sense (5′-TGGATCCGACATGTCAGA-3′) single-stranded oligonucleotide (both used at a final concentration of 1.5 μg/mL). Transfection was performed by using Lipofectamine reagent (Gibco, Milan, Italy) according to the manufacturer’s recommendations. Details of the Flip antisense and sense oligonucleotides have been described elsewhere.30Perlman H. Pagliari L.J. Georganas C. Mano T. Walsh K. Pope R.M. FLICE-inhibitory protein expression during macrophage differentiation confers resistance to fas-mediated apoptosis.J Exp Med. 1999; 190: 1679-1688Crossref PubMed Scopus (219) Google Scholar After 24 hours of treatment, LPMCs were incubated with CH11 (final concentration, 200 ng/mL) for a further 18 hours. Total proteins were extracted from whole mucosal biopsy specimens taken from 11 CD patients, 6 UC patients, and 11 normal controls. Proteins also were prepared from unfractionated LPMCs and purified CD3+, CD45RO+ LPLs, and CD3 and CD2-stimulated LPLs. Extracts were obtained using a buffer containing 10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, .4 mol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and 10% glycerol, 1 mmol/L dithiothreitol, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 1 mmol/L phenylmethanesulphonyl fluoride (all from Sigma). For the detection of FlipL/S, total proteins (80 μg/sample) were separated on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. A commercially available rat anti-human monoclonal Flip antibody recognizing both Flip L and S isoforms (clone DAVE-2; Alexis-Vinci, Florence, Italy; final concentration, 1 μg/mL) followed by a horseradish-peroxidase–conjugated rabbit anti-rat IgG antibody (DAKO, Glostrup, Denmark; final dilution, 1:10,000) was used, and the reaction was detected with a DURA chemiluminescence kit (Pierce, Rockford, IL). After detection of Flip, blots were stripped and incubated with a mouse anti-human β-actin antibody (Sigma; final dilution 1:5000) followed by a goat anti-mouse antibody conjugated to horseradish peroxidase (DAKO; final dilution, 1:20,000). IL-21 was assessed as previously described.35Monteleone G. Monteleone I. Fina D. Vavassori P. Del Vecchio Blanco G. Caruso R. Tersigni R. Alessandroni L. Biancne L. Naccari G.C. MacDonald T.T. Pallone F. Interleukin-21 enhances T-helper cell type I signaling and interferon-γ production in Crohn’s disease.Gastroenterology. 2005; 128: 687-694Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar Computer-assisted scanning densitometry was used to analyze the intensity of the immunoreactive bands. The percentage of Annexin-V–positive CD3+ LPLs was assessed by flow cytometry (FACSCalibur; Becton Dickinson, Milan, Italy). Briefly, at the end of the culture period, cells were washed twice in phosphate-buffered saline with 2% fetal calf serum and then incubated with anti-human CD3 fluorochrome-conjugated (FITC) monoclonal antibody (clone UCHT1; Beckman Coulter, Marseille, France) for 20′. Cells then were resuspended in 50 μL binding buffer solution (10 mmol/L HEPES/NaOH, pH 7.4, 140 mmol/L NaCl, 2.5 mmol/L CaCl2) containing 2.5 μL of phycoerithryn-conjugated Annexin-V (Becton Dickinson). Data were compared using the Student t test. To confirm that in CD mucosa LPLs are resistant to FAS-mediated apoptosis, normal, UC, and CD LPMCs either were left unstimulated or stimulated with CH11, and the percentage of apoptotic CD3+ LPLs then was evaluated by flow cytometry after 24 hours of culture. As expected, after CH11 stimulation, the rate of Annexin-V positivity was higher in normal (56.75% ± 4.9%) and UC (52% ± 9%) than CD (37.4% ± 3%) LPL samples (Figure 1), further corroborating previous studies.17Ina K. Itoh J. Fukushima K. Kusugami K. Yamaguchi T. Kyokane K. Imada A. Binion D.G. Musso A. West G.A. Dobrea G.M. McCormick T.S. Lapetina E.G. Levine A.D. Ottaway C.A. Fiocchi C. Resistance of Crohn’s disease T cells to multiple apoptotic signals is associated with a Bcl-2/Bax mucosal imbalance.J Immunol. 1999; 163: 1081-1090PubMed Google Scholar In addition, we evaluated the apoptosis of LPLs induced by CD2 pathway stimulation. As shown in Figure 1, stimulation with anti-CD3 and anti-CD2 enhanced the rate of apoptosis of normal and inflammatory bowel disease (IBD) LPLs. However, the mean apoptosis value of LPLs in CD (36.5% ± 3.5%, P = .03) was significantly lower than that seen in normal (45.5% ± 6.4%) and UC (41% ± 9%) LPLs (Figure 1). Because Flip plays a key role in preventing FAS-mediated T-cell apoptosis,10Thome M. Tschopp J. Regulation of lymphocyte proliferation and death by FLIP.Nat Rev Immunol. 2001; 1: 50-58Crossref PubMed Scopus (352) Google Scholar we then examined if the decreased rate of CD LPL apoptosis in response to FAS stimulation was associated with changes in the intracellular level of Flip. To address this issue, we first looked at the expression of Flip in freshly obtained whole biopsy specimens by Western blotting using an antibody that recognizes both the L and S isoforms of Flip. The isoform FlipL was detected in all mucosal samples regardless of whether biopsy specimens were taken from patients with CD, UC, or controls (Figure 2). However, immunoreactivity corresponding to FlipL was more pronounced in CD in comparison with UC and normal samples (Figure 2). Densitometric analysis of Western blot bands showed that the mean value of FlipL was significantly higher in patients with active CD (.79 ± .14 arbitrary units) than patients with UC (.57 ± .14, P < .01) and normal controls (.48 ± .17, P < .01) (Figure 2B). In addition, the isoform FlipS, whose expression confers a major resistance of T cells to FAS-mediated apoptotic signaling,36Schmitz I. Weyd H. Krueger A. Baumann S. Fas S.C. Krammer P.H. Kirchhoff S. Resistance of short term activated T cells to CD95-mediated apoptosis correlates with de novo protein synthesis of c-FLIPshort.J Immunol. 2004; 172: 2194-2200Crossref PubMed Scopus (72) Google Scholar was seen in 11 of 11 CD, 5 of 6 UC, and 3 of 11 normal control samples (Figure 2A and B). Densitometric analysis of Western blot bands showed that the mean value of FlipS was significantly higher in patients with active CD (.168; range, .09–.29 arbitrary units) than patients with UC (.07; range, 0–.17; P < .01) and normal controls (.02; range, 0–.15; P < .01) (Figure 2B). To examine if in CD tissue the expression of Flip correlates with the site of inflammation, paired biopsy specimens were taken from both inflamed and uninflamed mucosal areas of 6 patients with CD and examined for Flip. As shown in Figure 2C, a high expression of both FlipL and S isoforms was found only in biopsy specimens taken from inflamed sites in comparison with normal controls.Figure 2Flip 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because whole mucosal biopsy specimens contain several cell types that potentially could express Flip, we extended our analysis to purified CD3+ LPLs of CD and UC patients and controls. Expression of FlipL in CD3+ LPLs was higher in patients with CD (.66 ± .18 arbitrary units) than UC (.19 ± .19) and controls (.045 ± .08) (Figure 3A and B) (P < .001). Similarly, enhanced expression of FlipS was seen in patients with active CD (.23 ± .11) in comparison with UC patients (.04 ± .05) and normal controls (.02 ± .03) (P = .01) (Figure 3A and B). There is evidence that Flip levels can be down-regulated in T cells after activation.10Thome M. Tschopp J. Regulation of lymphocyte proliferation and death by FLIP.Nat Rev Immunol. 2001; 1: 50-58Crossref PubMed Scopus (352) Google Scholar This would thus seem to suggest that, in CD, high Flip levels may depend on the mucosal accumulation of naive T cells, which are recruited continuously from the peripheral blood.37Burgio V.L. Fais S. Boirivant M. Perrone A. Pallone F. Peripheral monocyte and naive T-cell recruitment and activation in Crohn’s disease.Gastroenterology. 1995; 109: 1029-1038Abstract Full Text PDF PubMed Scopus (126) Google Scholar To exclude this possibility, we analyzed Flip levels in CD45RO+ LPLs purified from CD patients and controls. Figure 3C clea" @default.
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• W1976779503 date "2006-02-01" @default.
• W1976779503 modified "2023-10-15" @default.
• W1976779503 title "A Functional Role of Flip in Conferring Resistance of Crohn’s Disease Lamina Propria Lymphocytes to FAS-Mediated Apoptosis" @default.
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