NO-mesalamine protects colonic epithelial cells against apoptotic damage induced by proinflammatory cytokines

Stefano Fiorucci1, Eleonora Distrutti1, Maureen N. Ajuebor2, Andrea Mencarelli1, Roberta Mannucci3, Barbara Palazzetti1, Piero Del Soldato4, Antonio Morelli1, and John L. Wallace2

1 Clinica di Gastroenterologia ed Epatologia and 3 Sezione di Medicina Interna e Scienze Oncologiche, Dipartimento di Medicina Clinica e Sperimentale, Università degli Studi di Perugia, 06100 Perugia, Italy; 2 Mucosal Inflammation Research Group, University of Calgary, Calgary T2N 4N1, Alberta, Canada; and 4 Nicox, Sophia Antipolis 06906, France


    ABSTRACT
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INTRODUCTION
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The activation of a self-amplifying cascade of caspases, of which caspase-8 is the apical protease, mediates Fas-, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-, and TNF-alpha -induced apoptosis in colon cell lines. Nitric oxide (NO) protects from apoptosis induced by Fas and TNF-alpha . We examined whether NCX-456, an NO-releasing derivative of mesalamine, protects colon epithelial cells from cytokine-induced apoptosis. Caco-2 and HT-29 cell lines express death factor receptors and are driven to apoptosis in response to incubation with Fas-agonistic antibody, TNF-alpha /interferon-gamma , and TRAIL. The two novel observations reported here are that 1) cotreatment of cells with NCX-456, but not mesalamine, resulted in concentration-dependent protection against death factor-induced apoptosis and inhibition of caspase activity, and 2) exposure to dithiothreitol, an agent that effectively removes NO from thiol groups, resulted in a 70% recovery of caspase activity, which is consistent with S-nitrosation as a major mechanism for caspase inactivation. These data suggest that caspase S-nitrosation represents a mechanism for protection of colonic mucosal epithelial cells from death factor-induced death.

colon cancer cells; apoptosis; death factors; nitromesalamine


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INTRODUCTION
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CROHN'S DISEASE and ulcerative colitis, the two main forms of inflammatory bowel disease (IBD), are chronic, spontaneously relapsing disorders that appear to be immunologically mediated (13). Although the underlying genetic and environmental causes remain to be elucidated, CD4+ T helper 1 (Th1) lymphocytes play a pivotal role in the pathogenesis of these diseases. A Th1-like phenotype, with its signature cytokines interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha , is shared among many colitis models and is found in patients with active Crohn's disease (6, 8, 13). The role of T cell-derived cytokines seems to be critical because IFN-gamma and TNF-alpha drive colonic epithelial cells to apoptosis and injection of anti-TNF-alpha antibodies in patients with active Crohn's disease leads to a transient improvement of the disease and attenuates the development of colitis in some experimental mouse models (13). Moreover, IFN-gamma not only cooperates with TNF-alpha to cause colonic cell death, it also modulates epithelial Fas expression and sensitizes colonic epithelial cells to Fas-induced apoptosis (13).

Apoptosis, or programmed cell death, is regulated by tightly controlled intracellular signaling events in response to pathological cytotoxic stimuli including TNF-alpha , TNF-related apoptosis-inducing ligand (TRAIL), and Fas (3, 4, 37). Fas, a transmembrane receptor that belongs to the TNF-alpha receptor family, is constitutively expressed by the basolateral membrane of normal colon and small intestinal epithelium (6, 8, 16, 22, 36, 38). Fas ligation induces apoptosis in colonic epithelial cells and is implicated in the epithelial damage seen in ulcerative colitis (6, 8, 16, 22, 36, 38). The ligand for Fas (FasL) is expressed by intraepithelial and lamina propria lymphocytes, and its expression is increased in the lamina propria of ulcerative colitis patients, suggesting that Fas-FasL-induced apoptosis participates in the mucosal damage of ulcerative colitis (6, 8). Inhibition of cytokine-regulated apoptosis may therefore be useful in preventing or treating intestinal lesions in patients with IBD.

One way of interfering with the progression of disease is to reduce intracellular events that lead to apoptotic cell death. The intracellular domains of Fas, TRAIL receptors 1 and 2 (TRAIL-R1 and -R2), and TNF-alpha receptor 1 (TNF-R1) contain a region termed the death domain (DD), which is required for induction of apoptosis (4, 37). Binding of Fas by FasL, TRAIL-R1 or -R2 by TRAIL, and TNF-R1 by TNF-alpha leads to the association of Fas-associated DD (FADD) and TNF-R1-associated DD (TRADD), respectively, with Fas, TRAIL-R1 and R2, or TNF-R1 via their homologous DD (4, 37). In their turn, FADD and TRADD recruit the zymogen form of the apoptosis-initiating protease, caspase-8, a member of the interleukin 1 (IL)-beta -converting enzyme (ICE) family (3, 37), through homophilic interaction of "death effector domains" leading to the assembly of a death-inducing signaling complex (DISC) at the cytoplasmic DD of Fas, TRAIL-R1, TRAIL-R2, or TNF-R1 (4). The proximity of caspase-8 zymogens facilitates activation through self-processing, leading to cleavage of downstream caspases including caspase-3, which is largely responsible, directly and indirectly, for dismantling the apoptotic cells from within (37). Inhibition of proapoptotic caspases is therefore a new pharmacological means of protection from death factor-induced apoptosis (11).

Mesalamine (5-aminosalicylic acid) is one of the most commonly used drugs for the treatment of active IBD and for maintenance of remission (34). However, the mechanism by which mesalamine reduces mucosal injury in colitis is not clear. Mesalamine exerts many effects that could contribute to its anti-inflammatory activity. These include inhibition of leukotriene synthesis, scavenging of oxygen-derived free radicals, scavenging of peroxynitrite, and inhibition of IL-1beta synthesis (34). Mesalamine has also been demonstrated to reduce peroxynitrite-induced apoptosis in Caco-2 cells, but whether it is also active in protecting from cytokine-induced apoptosis is unknown (33).

In recent years, we (14, 15) and others (12, 39) have demonstrated that the addition of a nitric oxide (NO)-releasing moiety to conventional drugs, such as aspirin and other anti-inflammatory analgesic drugs, and more recently to acetaminophen, results in new chemical entities that share the property of releasing small amounts of NO at target tissues. An increasing body of evidence demonstrates that the addition of the NO-releasing moiety to these compounds markedly enhances their anti-inflammatory, antipyretic, and/or analgesic effects (14, 15, 39, 40). Although the molecular mechanisms underlying the enhanced anti-inflammatory properties of NO-releasing compounds are poorly defined, we demonstrated previously (14, 15) that, similarly to NO, they cause the nitrosation/inhibition of cysteine proteases that mediate inflammation (ICE/caspase-1) and apoptosis (caspases-9 and -3). On the basis of these findings we hypothesized that adding an NO-releasing moiety to mesalamine would increase its effectiveness in reducing colonic inflammation. Indeed, we reported (40) that an NO-mesalamine derivative (hereafter referred to as NCX-456) exhibited improved efficacy over mesalamine in a rat model of chemically induced colitis. Compared with mesalamine, NCX-456 was more effective in reducing inflammation and chemotaxin-induced leukocyte adherence to the mesenteric endothelium and released NO when incubated with colonic mucosa fragments (40). However, NCX-456 failed to inhibit IFN-gamma release in the inflamed colon, suggesting that it may protect colon cells by acting downstream to this cytokine. Because protection against cytokine-induced apoptosis/necrosis contributes to increased drug efficacy, the present study was designed to investigate whether NCX-456 protects colonic epithelial cells against apoptosis induced by Fas, IFN-gamma , TRAIL, and TNF-alpha and to define the intracellular mechanism involved in this effect. Moreover, because it is still unknown whether colon cell lines express functionally active TRAIL receptors, we have evaluated the presence and functional activity of receptors for this death factor.


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Materials. RPMI-1640 medium, L-glutamine, EGTA, FCS, concanavalin A, aprotinin, leupeptin, dithiothreitol (DTT), HEPES, propidium iodide, BSA fraction V, phenylmethylsulfonyl fluoride (PMSF), and mesalamine were from Sigma Chemical (St. Louis, MO). Recombinant human IFN-gamma and TNF-alpha were from ICN Flow (Milan, Italy), whereas other media and serum were purchased from GIBCO (Paisley, UK). The anti-human Fas antibody (APO-1-3) and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) were from Alexis (San Diego, CA). The anti-human caspase-8 antibody (p20 subunit) was from New England Biolab (Beverly, MA), and anti-human polyclonal cytochrome c (Cyt c) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). TRAIL peptide was from Biomol (Plymouth Meeting, PA), and 4,5-diaminofluorescein diacetate (DAF-DA) was from Calbiochem (Darmstadt, Germany). NO-mesalamine [NCX-456; 5-amino-2-hydroxybenzoic acid 4-(nitroxybutylester)] was synthesized by Nicox (Nice, France).

Cell culture. HT-29 and Caco-2, two human colon adenocarcinoma cell lines, were obtained from American Type Culture Collection (Rockville, MD). HT-29 cells were cultured in RPMI-1640 medium containing 10% FCS, glutamine (2 mM), and antibiotics (50 U/ml penicillin, 50 µg/ml streptomycin) (1, 2). Caco-2 cells were cultured in MEM containing 20% FCS, glutamine, and antibiotics and supplemented with nonessential amino acids and 1 mM sodium pyruvate. Both types of cells were grown in 75-cm2 culture flasks, and the medium was changed every other day. The cells were passaged weekly and, for experiments, were aliquoted into 96-well plates (104 cells/well) and allowed to adhere overnight before cytokine stimulation in medium without serum.

Induction and detection of apoptosis. To test whether cytokines and death factors drive colon cells to apoptosis, HT-29 and Caco-2 cells were incubated with medium alone or IFN-gamma (1,000 U/ml) and TNF-alpha (100 ng/ml) or 10 µg/ml of APO-1-3 monoclonal anti-human Fas antibody or human recombinant TRAIL (100 ng/ml) alone or in combination for 8 h. In experiments in which IFN-gamma and Fas monoclonal antibody were used to induce apoptosis, cells were preincubated for 3 h with IFN-gamma and then Fas monoclonal antibody was added. To test whether NCX-456 and mesalamine modulate cytokine-induced apoptosis, cells were incubated with the cytokine mixture (i.e., IFN-gamma  + Fas agonistic monoclonal antibody or IFN-gamma  + TNF-alpha at the concentration described) with or without 1-100 µM NCX-456 or mesalamine for 8 h. In some experiments, cells were incubated with the NO scavenger PTIO (100 µM) to assess whether NO scavenging reversed the protective effect of NCX-456 on cytokine-induced cell death (3, 4). At the end of the culture period, adherent cells were detached and mixed with floating cells by gentle centrifugation. Apoptosis was detected by staining the cells with propidium iodide (PI). Briefly, cell pellets were washed twice in PBS, resuspended in hypotonic fluorochrome solution (50 µg/ml PI in 0.1% sodium citrate + 0.1% Triton X-100), kept 4-8 h at 4°C in the dark, and analyzed using a Epics XL flow cytometer (Beckman-Coulter, Miami, FL). The percentage of apoptotic cells was determined by evaluating hypodiploid nuclei after proper gating on DNA content. Alternatively, cells were recovered from cultures (both floating and adherent), fixed with 3.7% paraformaldehyde-PBS, and stained with 0.1 µg/ml 4,6-diamino-2-phenylindole (DAPI). The percentage of cells with condensed chromatin and fragmented nuclei was determined by ultraviolet (UV) microscopy (14). Each experiment was performed in triplicate on separate days.

Detection of apoptosis by ELISA assay. ELISA is based on the photometric sandwich-enzyme-immunoassay principle and uses mouse monoclonal antibodies directed against cytosolic DNA fragments and histones. Briefly, three incubation steps were performed. First, the antihistone antibody was fixed on the wall of a microtiter plate, Second, the nucleosomes contained in the sample were bound via their histone to the antihistone antibody. Third, anti-DNA-peroxidase was added to react with the DNA part of the nucleosome. The amount of peroxidase retained in the sample was determined photometrically (absorbance at 405/490 nm) with 2,2-azino-di-(3-ethylbenzthiazoline sulfonate) as a substrate (14, 15). Assays were performed using a cell death detection ELISA kit following the recommendations of the manufacturer (Boehringer Mannheim, Indianapolis, IN).

DNA fragmentation assay. In some experiments apoptosis was also assessed by DNA laddering assay, as previously described (14). Briefly, the floating cells and detached cells were collected and sedimented by centrifugation. Washed cell pellets were resuspended in cell lysis buffer [10 mM Tris · HCl (pH 7.4), 10 mM EDTA (pH 8.0), 0.5% Triton X-100] and incubated. RNase A (0.5 mg/ml) and proteinase K (0.5 mg/ml) were added, respectively, and incubated for 2 h. DNA was precipitated by ethanol, and 3 µg (or 1 × 106 cells) of water-diluted sample was run on a 2% agarose gel. Gels were stained with ethidium bromide, and DNA was visualized by UV transilluminator.

RT-PCR. Total RNA was isolated from human biopsies using TRIzol reagent (Life Technologies, Milan, Italy). First-strand cDNA was synthesized from total cellular RNA (1 µg) with 200 U of SuperScript II (Life Technologies), 500 ng of oligo(dT)15 primer (Sigma Genosys, Gallarate, Italy), and dNTPs (200 µM; Promega). Ten microliters of sample and two microliters of oligo(dT) primer were heated to 94°C for 2 min and cooled on ice for 5 min. To this mixture was added 4 µl of 5× first-strand buffer [250 mM Tris · HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2], 2 µl of DTT (0.1 M), and 1 µl of mixed dNTPs (10 mM), and the mixture was incubated at 42°C for 2 min. One microliter of SuperScript II then added, and the reaction was incubated at 42°C for 50 min, heated at 70°C for 10 min to inactivate the enzyme, and cooled at 4°C.

Multiplex PCR was performed with a Human Apoptosis Genes Set-3 kit (Maxim Biotech, San Francisco, CA) using human Fas gene, human Fas ligand gene, human TRAIL gene, human FLICE gene (caspase-8), and human GAPDH gene as a control. PCR was performed using specific primers (Sigma Genosys). For human TRAIL R-1 the sense primer was 5'-CGCACGAACTCAGCCAACGATT-3' and antisense was 5'-TTTCCACAGTGGCATTGGCACC-3'; for human TRAIL R-2 the sense primer was 5'-CAAAATACACCGACGATGCCCG-3' and antisense was 5'-GTGCAGCGCAAGCAGAAAAGGA-3'; for human TRAIL R-3 the sense primer was 5'-CGCACGAACTCAGCCAACGAT T-3' and antisense was 5'-CGACGATGACGACGACGAACTT-3'; for human TRAIL R-4 the sense primer was 5'-TGGGACTTTGGGGCAAAAGCG T-3' and antisense was 5'-AGCAGAACCGCGACGATGAAGA-3'. The cDNA was amplified with a "hot start" reaction in a 20-µl reaction containing 5 µl of cDNA product, 2 µl of PCR buffer [200 mM Tris · HCl (pH 8.4), 500 mM KCl], 200 µM dNTPs, 1 µM sense and antisense primers, 1.5 mM MgCl2, 1 U of Platinum Taq polymerase (Life Technologies), and water in a Hybaid PCR Sprint thermocycler (Celbio, Milan, Italy). PCR was carried out for 35 cycles (30 min for amplification of beta -actin) as follows: 94°C for 30 s, 60°C for 15 s, and 72°C for 30 s, with a final extension at 72°C for 5 min. Multiplex PCR was carried out for 35 cycles as follows: 94°C for 1 min and 60°C for 1 min with a final extension at 72°C for 10 min. PCR products were then separated on 1.5% agarose gel and stained with 0.5 µg/ml ethidium bromide. The size of PCR products was assessed by comparison with 1 µg of 100-bp DNA ladder (Life Technologies). The gel was photographed under UV transillumination with a Kodak Digital Science ID Image Analysis Software (Kodak, Rochester, NY); images were then digitalized and a semiquantitative analysis was performed with the same software. Each assay was carried out in triplicate. The beta -actin primers were used as a control for both reverse transcription and the PCR reaction itself and also for comparing the amount of products from samples obtained with the same primer.

Assessment of caspase activity. After incubation with appropriate agents (see Fig. 2) HT-29 and Caco-2 cells were recovered into lysis buffer [10 mM Tris · HCl (pH 7.3), 25 mM NaCl, 0.25% Triton X-100, 1 mM EDTA]. After centrifugation at 16,000 g for 30 min, the resulting supernatants were adjusted to 1 mg/ml with lysis buffer and 25 µg total protein was incubated in 100 µl of caspase buffer [50 mM HEPES (pH 7.2), 100 mM NaCl, 1 mM EDTA (pH 8.0), 10% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 1 mM DTT] with various fluorogenic substrate peptides (100 µM) including acetyl-Asp-Glu-Val-Asp-(7-amino-4-trifluoromethyl-coumarin) (Ac-DEVD-AFC) for caspase-3, Ac-Ile-Glu-Thr-Asp-AFC (Ac-IETD-AFC) for caspase-8, and Ac-Leu-Glu-His-Asp-AFC (Ac-LEHD-AFC) for caspase-9. Caspase-8, -3, and -9 activity was assayed using a fluorimeter plate reader in kinetic mode with excitation and emission wavelengths of 405 and 519 nm, respectively, continuously measuring release of AFC from substrate peptides as previously described (14, 15)

Western blot analysis of caspase-8 cleavage. Cells (1 × 107) were lysed in 100 µl of lysis buffer [20 mM Tris · HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 1 mM PMSF, 40 µg/ml aprotinin, 20 µg/ml leupeptin] at 4°C. Ten microliters of sample were then run on 8-16% linear gradient polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA). Gels were transferred to nitrocellulose membrane (Hybond-C Extra, Amersham). Membranes were blocked in 5% milk powder in PBS and probed with either anti-FLICE antibody (M2, Sigma Chemical) or a rabbit polyclonal anti-caspase-8 antibody, both at dilutions of 1:1000. The secondary antibody was goat anti-mouse IgG horseradish peroxidase-conjugated antibody (PharMingen, San Diego, CA) used at a dilution of 1:1,000.

Analysis of Cyt c release. Caco-2 cells were scraped off in isotonic isolation buffer (in mM: 1 EDTA, 10 HEPES, 250 sucrose, pH 7.6), collected by centrifugation at 2,500 g for 5 min at 4°C, and resuspended in hypotonic isolation buffer (in mM: 1 EDTA, 10 HEPES, 50 sucrose, pH 7.6). Cells were then incubated at 37°C for 5 min and homogenized under a Teflon pestle. Hypertonic isolation buffer (in mM: 1 EDTA, 10 HEPES, 450 sucrose, pH 7.6) was added to balance the buffer's tonicity. Samples were centrifuged at 2,000 g for 5 min at 4°C. Supernatants were recovered and centrifuged again at 10,000 g for 10 min. The pellet contained the mitochondrial fraction, which was resuspended in isotonic isolation buffer, whereas the supernatant contained cytosolic proteins. Protein concentrations in pellets and supernatants were determined using the Bio-Rad protein assay kit according to the manufacturer's specifications. After electrophoresis separation of 50 µg of protein in sodium dodecyl sulfate-12% polyacrylamide, gels were transferred by semidry transfer (Bio-Rad) to nitrocellulose membranes. Immunoblots were blocked in TTBS [10 mM Tris · HCl, 150 mM NaCl (pH 7.5), 0.05% Tween 20] containing 5% nonfat dried milk and incubated overnight with the anti-Cyt c antibody (diluted 1:1,000 in TTBS with 0.5% nonfat dried milk). After being washed, membranes were incubated with peroxide-conjugated secondary antibody (1:5,000 in TTBS with 0.5% nonfat dried milk) for 2 h, and the blot was developed with the ECL system (ECL Western blotting kit, Amersham International).

Detection of intracellular NO formation. To assess whether colon cells generate intracellular NO or NO-derived compounds from NCX-456, adherent Caco-2 cells were incubated with 100 µM NCX-456, mesalamine, or S-nitroso-DL-penicillamine (SNAP) and intracellular NO formation was assessed according to the method of Nakatsubo et al. (30) with DAF-DA. Briefly, adherent cells (1 × 106/ml) were preincubated with 1 mM L-N6-(1-iminoethyl)lysine (L-NIL) for 30 min to suppress endogenous NO generation and then loaded by incubating them in PBS in the presence of 10 µM DAF-DA at 37°C for 30 min. Cells were then washed three times with PBS and placed on a confocal microscope (Bio-Rad 1024, Bio-Rad, Milan, Italy), and images were taken every 5 s as previously described (30). Image analysis was carried out using the LaserSharp software (Bio-Rad). Intracellular NO formation was also assessed by fluorimetry in detached Caco-2 cells. Briefly, after detachment, cells (1 × 106) were preincubated with 1 mM L-NIL for 30 min, loaded with 10 µM DAF-DA, and then added to a quartz cuvette with continuous stirring and temperature thermostatically maintained at 37°C with a Hitachi 2000 (Hitachi, Milan, Italy) fluorescence spectrophotometer. SNAP, NCX-4016, or mesalamine (100 µM) was then added to the cell suspension. Cells were excited at 395-nm wavelengths with a 10-nm slit, and the intensity of fluorescence emitted at 515 nm was recorded. NO generation was expressed in arbitrary units of absorbance (16).

Nitrite/nitrate assay. Nitrite/nitrate concentrations in cell supernatants were measured by a fluorimetric detection kit (Cayman Chemical, Ann Arbor, MI). The lower detection limit, as reported by the manufacturer, was ~4 pM/well (16).

Assessment of caspase S-nitrosation. To investigate whether the inhibition of caspaselike proteases exerted by NCX-456 was caused by protein S-nitrosation, cell lysates obtained from Caco-2 cells were incubated alone or with a cytokine mixture in combination with 100 µM NCX-456, mesalamine, or SNAP and were exposed to DTT (20 mM) to remove thiol-bound NO. The DTT and excess of NO were then removed by passing the sample through a Sephadex G-25 column preequilibrated with the lysis buffer and enzyme activity was assessed (14-16, 24-28). In another set of experiments, lysates obtained from Caco-2 cells incubated with Fas or IFN-gamma /TNF-alpha alone or in combination with NCX-456 were exposed to DTT (20 mM) and/or HgCl2 (5 mM) for 40 min on ice and caspase-8-like activity was measured (14-16, 24-28).

Data analysis. All values are expressed as means ± SE of n observations. Groups of data were compared using a one-way analysis of variance followed by a Student-Newman-Keuls test. With all analyses, an associated P value of <5% was considered significant (5).


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Death receptor expression on colon cancer lines. We first investigated whether HT-29 and Caco-2 cells express death receptors. As illustrated in Fig. 1, RT-PCR analysis demonstrated that Fas and TNF-R1 are expressed in both cell lines, although to a different extent, whereas TRAIL was not expressed. Both cell lines also expressed FLICE (caspase-8). As illustrated in Fig. 1B, both colon cancer cell lines expressed TRAIL receptors (R1-R4). Incubation of the cells with IFN-gamma markedly upregulated Fas mRNA expression in both cell lines, although it had no effect on TNF and TRAIL receptors (data not shown). Thus colon cell lines express death receptors.


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Fig. 1.   RT-PCR analysis of death factor receptor expression on colon cancer cell lines. A: multiple RT-PCR demonstrating the expression of Fas, FLICE (caspase-8), tumor necrosis factor (TNF)-alpha receptor 1 (TNF-R1), and TNF-related apoptosis-inducing ligand (TRAIL) on Caco-2 and HT-29 colon cancer cell lines. Lane 1, negative control; lane 2, positive control; lane 3, HT-29 cells; lane 4, Caco-2 cells. The blot is representative of at least 3 RT-PCR analyses. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B: Caco-2 cells express TRAIL receptors. For each blot: lane 1, molecular markers; lane 2, negative control; lane 3, positive control; lane 4, Caco-2 cells. Each blot is representative of at least 3 RT-PCR analyses.

Cytokines and Fas drive colon epithelial cells to apoptosis and require caspase activation. Incubation of growth-arrested HT-29 and Caco-2 monolayers with a combination of IFN-gamma (1000 U/ml) and TNF-alpha (100 ng/ml) or Fas-agonistic monoclonal antibody for 8 h resulted in a 30-35% increase in the expression of apoptotic markers, as assessed by PI incorporation, nuclei staining with DAPI, DNA fragmentation, and DNA-histone association (Figs. 2 and 3). In contrast, treatment with individual cytokines or a Fas-agonistic monoclonal antibody alone did not increase cell death above basal levels (Fig. 2). However, there were differences in the sensitivity of the two cell lines to apoptosis induced by the cytokine mixture, because HT-29 cells were sensitive to Fas- and TNF-alpha -induced apoptosis and Caco-2 cells were resistant to apoptosis induced by the Fas-agonistic monoclonal antibody. In contrast, both cell lines were sensitive to TRAIL-induced apoptosis (Fig. 2, B and C). In both cell lines morphological changes suggestive of apoptosis were associated with an increased activity of caspase-8 like proteases (Fig. 2C) as well as caspase-3- and -9-like proteases (data not shown). As shown in Fig. 2D, pretreatment of the cells with 100 µM Z-VAD-FMK, a pancaspase inhibitor, rescued both cell lines from apoptosis induced by IFN-gamma /TNF-alpha , IFN-gamma /Fas-agonistic antibody, and TRAIL and caused an ~80% reduction of caspase-8 activation caused by proapoptotic agents. Thus caspase activation is required for apoptotic signal transduction in colon cell lines challenged with TNF-alpha or Fas in the presence of IFN-gamma and TRAIL.


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Fig. 2.   A: structure of 5-amino-2-hydroxybenzoic acid 4-nitroxybutylester (NCX-456) and mesalamine. B: interferon (IFN)-gamma sensitized HT-29 and Caco-2 cells to TNF-alpha and Fas-induced apoptosis. HT-29 and Caco-2 cells (106 cells/ml) were either left untreated (control) or pretreated with IFN-gamma (300 U/ml) for 24 h and then exposed to TRAIL, TNF-alpha , or APO-1-3, a Fas agonistic antibody (Ab), for 8 h. At the end of incubation, the supernatants were removed, cell pellets were lysed, and apoptosis was determined by staining the cells with propidium iodide (PI) and assaying the number of apoptotic/necrotic cells at flow cytometry as described in MATERIALS AND METHODS. Data are means ± SE of 6 experiments. *P < 0.01 vs. control cells. C: death factor-induced apoptosis is associated with caspase-8 activation. Data are means ± SE of 6 experiments. *P < 0.01 vs. control cells. D: effect of the pancaspase inhibitor Z-VAD-FMK on cytokine-induced apoptosis. HT-29 and Caco-2 cells were either left untreated or treated with 50 µM Z-VAD-FMK for 1 h. Where indicated, the cells were then further treated with combinations of IFN-gamma  + APO-1-3 (100 ng/ml) (HT-29) or IFN-gamma  + TNF-alpha (Caco-2) for 8 h. The number of apoptotic/necrotic cells at flow cytometry was assessed by flow cytometry. Data are means ± SE of 6 experiments. *P < 0.01 vs. control cells; **P < 0.01 vs. cells treated with the cytokine mixture.



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Fig. 3.   4,6-Diamino-phenylindole (DAPI) staining of nuclei from Caco-2 cells. A: control cells. B: cells treated with IFN-gamma and TNF-alpha for 8 h. C: cells treated with cytokine mixture + NCX-456 (100 µM). D: cells treated with the cytokine mixture + mesalamine (100 µM). A-D are representative of at least 3 other experiments. E: cytokine treatment causes cytochrome c (Cyt c) release. Caco-2 cells were incubated with cytokine mixture with or without NCX-456 or mesalamine, and Cyt c content in the cytosol and mitochondrial fraction was assayed (see MATERIALS AND METHODS). Top, Cyt c content in mitochondria; bottom, Cyt C content in cytosol of Caco 2 cells. Lane 1, control; lane 2, cells treated with cytokine mixture; lane 3, cells treated with cytokines + mesalamine; lane 4, cells treated with cytokines + NCX-456. F: effect of NCX-456 or mesalamine on DNA fragmentation. Lane 1, molecular markers; lane 2, control cells; lane 3, cells incubated with the cytokine mixture; lane 4, cells incubated with the cytokine mixture + NCX-456; and lane 5, cells incubated with the cytokine mixture + mesalamine. G: DNA fragmentation analysis of Caco-2 cells incubated with cytokine mixture with or without NCX-456 or mesalamine. DNA fragmentation was assessed by an ELISA that specifically detects histone-associated DNA. *P < 0.01 vs. control cells; **P < 0.01 vs. cells treated with the cytokine mixture.

NCX-456 protects colon epithelial cells against cytokine-induced apoptosis. Exposure of HT-29 and Caco-2 cells to NCX-456, but not mesalamine, protected against cytokine-induced cell death (Figs. 3 and 4). Indeed, at a concentration of 100 µM, the NO-mesalamine derivative caused ~80% reduction of death factor-induced lethality (P < 0.001 vs. cytokine alone) as measured by PI incorporation. Mesalamine (100 µM) also caused ~30% reduction of apoptosis caused by a combination of IFN-gamma with Fas or TNF-alpha , although it was significantly less effective than NCX-456 (P < 0.001). As shown in Fig. 4, the protective effect exerted by NCX-456 was concentration dependent and lasted for at least 24 h. Protection exerted by NCX-456 was detectable at a concentration of 1 µM, half-maximal at 6 µM, and maximal at 100 µM. Coincubation of Caco-2 cells with NCX-456 (100 µM) was also effective in preventing changes of nuclear morphology as evaluated by DAPI staining and DNA fragmentation as assessed by DNA ladder assay and DNA-histone formation (Fig. 3). Moreover, as shown in Fig. 3E, NCX-456 inhibited cytokine-induced Cyt c release from mitochondria. Mesalamine was significantly less effective than NCX-456 in reducing these markers of apoptosis (Fig. 3), and at a concentration of 100 µM it had no effect on DNA fragmentation or Cyt c release.


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Fig. 4.   NCX-456 protects from cytokine-induced cell death. A: epithelial colon cell lines were incubated with the cytokine mixture, as in Fig. 1, with or without 100 µM NCX-456 or mesalamine for 8 h. Cell death was assessed by flow cytometry after cell staining with PI. Data are means ± SE of 4-6 experiments. *P < 0.01 vs. control cells; **P < 0.01 vs. cells treated with cytokine mixture. B: NCX-456 exerts dose-dependent protection against damage induced by IFN-gamma /Fas in HT-29 cells. Data are means ± SE of 4-6 experiments. *P < 0.01 vs. control cells. C: protection exerted by NCX-456 is maintained over time. HT-29 cells were incubated with the cytokine mixture, as in Fig. 1, with or without 100 µM NCX-456 or mesalamine up to 24 h, and cell death rate was assessed at different time points. Data are means ± SE of 4-6 experiments. *P < 0.01 vs. cells treated with cytokine mixture.

Inhibition of apoptosis caused by NCX-456 is mediated by an NO-dependent mechanism. To gain insight on the mechanism responsible for apoptosis inhibition, we then evaluated whether incubating the cells with an NO scavenger reversed the protection exerted by NCX-456. As shown in Fig. 5, the ability of NCX-456 to rescue cells from death induced by IFN-gamma /TNF-alpha , IFN-gamma /FasL, and TRAIL was significantly reduced when the NO scavenger PTIO (100 µM) was added to the incubation medium. Thus NCX-456 acts through an NO-dependent pathway.


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Fig. 5.   2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), a NO scavenger, reverses protection exerted by NCX-456. A: effect of PTIO on protection exerted by NCX-456 against cell death induced by FasL and TNF-alpha in IFN-gamma -treated cells. Cell death was assessed by PI staining (see MATERIALS AND METHODS). Data are means ± SE of 6 experiments. *P < 0.01 vs. control cells; ** P < 0.01 vs. cells treated with cytokine alone; ***P < 0.01 vs. cells treated with cytokine + NCX-456. B: PTIO reverses protection exerted by NCX-456 against TRAIL-induced apoptosis. Cell death was assessed by PI staining. Data are means ± SE of 6 experiments. *P < 0.01 vs. control cells; ** P < 0.01 vs. cells treated with TRAIL alone; ***P < 0.01 vs. cells treated with TRAIL + NCX-456.

NCX-456 inhibits caspase activity. Because activation of the caspase cascade is pivotal to the death execution phase of apoptosis, we tested the effect of NCX-456 on proapoptotic caspases. As illustrated in Fig. 6, incubation of Caco-2 (Fig. 6, A-C) and HT-29 (data not shown) cells with 100 µM NCX-456 resulted in a 70-80% reduction of cytokine-induced caspase-8, -9, and -3 activity. To confirm that NCX-456 directly inhibits caspase-8 activation, we examined the cleavage of the procaspase-8 zymogen by Western blotting with an antibody that specifically detects the p20 subunit of this caspase. After treatment with IFN-gamma /TNF-alpha , procaspase-8 was cleaved in Caco-2 and HT-29 cells. Cotreatment of the cells with NCX-456, but not with mesalamine, almost completely abolished procaspase-8 cleavage (Fig. 6D). Thus NCX-456 protects colon epithelial cell against cytokine-induced apoptosis by inhibiting proapoptotic caspases.


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Fig. 6.   NCX-456 inhibits caspase activation. A-C: NCX-456 (100 µM) inhibits caspase activation in HT-29 cells. Data are means ± SE of 6 experiments. *P < 0.01 vs. control cells; **P < 0.01 vs. cells treated with cytokine mixture. D: NCX-456, but not mesalamine, inhibits procaspase-8 cleavage. Western blot analysis of procaspase-8 cleavage induced by IFN-gamma and Fas is shown. Lane 1, control cells; lane 2, cells treated with cytokine mixture; lane 3, cells treated with cytokine + NCX-456; lane 4, cells treated with cytokine + mesalamine. Figure is representative of 3 others.

NCX-456 inhibits caspase activity by nitrosation. The association between the suppression of cytokine-induced apoptosis and caspase inhibition suggests that NCX-456 inhibits apoptosis by blocking caspase activation. Because there is evidence that NO-releasing compounds inhibit caspase activities by causing enzyme nitrosation, we next investigated biochemically whether exposure to NCX-456 results in caspase-8 nitrosation/inhibition. To ascertain this point, lysates obtained from cytokine-treated cells were incubated with 20 mM DTT, an agent that effectively displaces thiol-bound group from proteins. As shown in Fig. 7, exposure to DTT resulted in ~70% recovery of protease activity in lysates obtained from cells incubated with cytokine plus NCX-456. Confirming the role of NO in the inhibition of caspase-8, SNAP (100 µM) caused 80% reduction of caspase-8-like activity induced by TNF-alpha and IFN-gamma (Fig. 7A), an effect that was significantly reduced by DTT (P < 0.01). Caspase-8-like activity was also reduced by incubating lysates obtained from cytokine-treated cells with HgCl2, an agent that binds thiol groups and removes NO. Indeed, at a concentration of 5 mM, HgCl2 caused a 90% loss of proteolytic activity (Fig. 7B). Inhibition induced by HgCl2 was partially reversed by DTT. Thus NCX-456 causes caspase nitrosation.


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Fig. 7.   NCX-456 causes caspase-8 S-nitrosation. A: dithiothreitol (DTT), an agent that removes thiol-bound nitric oxide (NO), causes a 70% recovery of caspase-8 activity in HT-29 cell lysates. Cells were treated with the cytokine mixture alone or cytokine + 100 µM NCX-456, mesalamine, or S-nitroso-DL-penicillamine (SNAP). After 8 h of incubation cell lysates were prepared and incubated with 20 mM DTT. Data are means ± SE of 6 experiments. *P < 0.01 vs. control; **P < 0.01, DTT vs. cytokine mixture + NCX-456; ***P < 0.01, NCX-456 or SNAP vs. cells treated with cytokine mixture. B: SNAP causes caspase-8 S-nitrosation. Data are means ± SE of 6 experiments. *P < 0.01 vs. control cells; **P < 0.01, DTT vs. cytokine mixture + SNAP; ***P < 0.01, SNAP-treated cells vs. cells treated with cytokine mixture.

NCX-456 causes intracellular NO formation. Because these data indicate that NCX-456-derived NO was responsible for the antiapoptotic effect exerted by NCX-456, we next investigated whether incubation of Caco-2 cells with this compound results in intracellular NO formation. As shown in Fig. 8, exposure of DAF-DA-loaded cells to NCX-456 (100 µM) resulted in a time-dependent increase in intracellular fluorescence, suggesting that NCX-456 penetrates colon cell membranes and is metabolized to release NO. The cell metabolism, however, is slow, because maximal fluorescence was evident after 3 h of incubation. In contrast to NCX-456, the addition of SNAP caused a rapid increase in intracellular fluorescence (Fig. 8D). No changes in intracellular fluorescence were documented in cells incubated with mesalamine alone (data not shown). To confirm these findings by another means, we measured intracellular NO fluorescence in nonadherent Caco-2 cells incubated with 100 µM NCX-456, SNAP, or mesalamine. As illustrated in Fig. 8E, although exposure of the DAF-DA-loaded cells to SNAP resulted in a rapid rise of intracellular fluorescence, NCX-456 caused a slow but sustained increase of intracellular fluorescence. Again, no changes were measured in cells loaded with mesalamine. In confirmation of these findings, incubation of the cells with SNAP or NCX-456 resulted in a significant increase in nitrite/nitrate release (Fig. 8F). Exposure of Caco-2 cells to 100 µM NCX-456 resulted in a high output of nitrite/nitrate in cell supernatants (~150 µM/107 cells at 3 h of incubation). No changes in nitrite/nitrate production were observed in cells incubated with mesalamine alone (P < 0.001 compared with NCX-456). Thus NCX-456 is metabolized by colon epithelial cells to release intracellular NO.


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Fig. 8.   Time course of intracellular NO formation in 4,5-diaminofluorescein diacetate (DAF-DA)-loaded Caco-2 cells exposed to NCX-456. Cells were incubated with no agent (A) or with 100 µM NCX-456 for 1 and 3 h (B and C), and intracellular fluorescence was recorded by confocal microscopy; original magnification, ×400. Inset C1, higher magnification of cells demonstrating subcellular distribution of NO formation in Caco-2 cells; original magnification, ×600. D: effect of SNAP on intracellular fluorescence in DAF-DA-loaded Caco-2 cells. Cells were incubated with 100 µm SNAP for 5 min. E: SNAP causes a rapid increase in intracellular fluorescence in nonadherent Caco-2 cells. Cells were incubated for different amounts of time with 100 µM SNAP, NCX-456, or mesalamine, and intracellular fluorescence was continuously assessed by fluorimetry. F: nitrite/nitrate release in cell supernatants. Caco-2 cells were incubated with no agent or with 100 µM NCX-456, mesalamine, or SNAP, and nitrite/nitrate concentrations in cell supernatants assessed were as indicated in MATERIALS AND METHODS. Data are means ± SE of 6 experiments. *P < 0.01 vs. control cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

T lymphocyte-derived cytokines not only recruit inflammatory cells at the site of inflammation but directly kill colonic epithelial cells or sensitize them to death induced by death factors such as TNF-alpha and FasL. TNF-alpha and FasL are expressed by intraepithelial and lamina propria lymphocytes, and their expression is increased in the lamina propria of ulcerative colitis patients (6, 8, 13). The effects of T cell-derived cytokines on cultured intestinal epithelial cells have also been well documented because human lamina propria T lymphocytes produce IFN-gamma and TNF-alpha , which act in concert to kill colon epithelial cells. The effects of IFN-gamma on colon epithelial cells may have clinical relevance, as this cytokine has been implicated in a variety of inflammatory conditions including ulcerative colitis, Crohn's disease, and human immunodeficiency virus-related enterocolitis (13). Indeed, biological agents that inhibit TNF-alpha and/or IFN-gamma production (IL-10 and thalidomide) or TNF-alpha activity (infliximab) effectively reduce inflammation in IBD patients (31, 34). In the present study we demonstrated that exposure of two cell lines, which express different stages of epithelial cell maturation, to IFN-gamma sensitizes them to the proapoptotic/necrotic effect of a Fas-agonistic monoclonal antibody and TNF-alpha and that this effect is mediated by activation of the caspase cascade. However, our results demonstrated that Caco-2 and HT-29 cells have different sensitivities to the apoptosis induced by death factors, because Caco-2 cells, in contrast to HT-29 cells, are resistant to Fas-mediated apoptosis (1, 2). In line with this finding, it was demonstrated previously (1, 2) that Caco-2 cells not only express very low amounts of Fas but lack one or more presently unidentified factor(s) necessary to activate the chain of reactions that leads to caspase-8 recruitment. In the present study we also demonstrated for the first time that colon cancer lines express TRAIL receptors and are sensitive to TRAIL-induced apoptosis (4). These cells are therefore a useful model for the study of the effect of drugs on cytokine-regulated apoptosis in colonic mucosa.

NCX-456 is a derivative of mesalamine that consists of the parent drug linked to an NO-releasing moiety through an ester linkage. In a previous study (40) we demonstrated that this compound was significantly more effective than mesalamine in reducing colonic inflammation in a rat model of colitis. In the present study we have extended these observations by demonstrating that NCX-456 directly protects IFN-gamma -sensitized colon epithelial cells from apoptotic/necrotic death induced by TNF-alpha or Fas-agonistic monoclonal antibody by causing the S-nitrosation/inhibition of proapoptotic caspases. Activation of the caspase cascade appears to be essential to cytokine-induced apoptosis of HT-29 and Caco-2 cells, in light of our observation that pretreatment with the pancaspase inhibitor Z-VAD-FMK completely prevents apoptosis induced by TNF-alpha and Fas (Fig. 2). Our current understanding of caspase involvement in Fas/TNF-alpha -induced apoptosis indicates that Fas and/or TNF-R1 cross-linking leads to the activation of a self-amplifying cascade of caspases, of which caspase-8 is the apical protease (37). This particular caspase is recruited and probably auto-proteolytically activated through protein-protein interaction with FADD among a complex of proteins (DISC) that dock onto the cytoplasmic DD of oligomerized Fas. Activated caspase-8 releases active caspase-8 subunits that can then cleave other caspases (e.g., caspase-3), which can in turn cleave a large number of intracellular death substrates. In the present study, we demonstrated that, in contrast to mesalamine, NCX-456 prevents caspase activation in Fas- and TNF-alpha -challenged cells and that this effect is mainly mediated by protein nitrosation/inhibition. Supporting this view, we demonstrated that 1) caspases-8, -3, and -9 are inhibited in NCX-456-treated cells but not in cells treated with mesalamine, which suggests that the NO group of NCX-456 is responsible for this effect; 2) exposure to PTIO, an NO scavenger, reversed protection exerted by NCX-456 on cytokine-induced apoptosis (Fig. 5); 3) exposure of NCX-456-treated cells to DTT, an agent that effectively removes NO from thiol groups, resulted in a 70% recovery of caspase activity, which is consistent with nitrosation as a major mechanism for caspase inhibition; 4) HgCl2, which binds thiol groups, caused a DTT-reversible inhibition of caspase activity, again suggesting a role for protein nitrosation in caspase inhibition (14-16, 24-26); 5) similarly to NCX-456, the NO donor SNAP inhibited caspase activity and rescued cells from death induced by Fas and TNF-alpha ; and 6) incubation of nontreated Caco-2 cells with NCX-456 resulted in a time- and concentration-dependent increase in intracellular NO as detected by intracellular fluorescence in adherent and nonadherent cells and nitrite/nitrate in cell supernatants. In contrast to NCX-456, mesalamine did not cause NO formation and was significantly less effective than NCX-456 in protecting from apoptosis caused by Fas and TNF-alpha (33). Together these data suggest that NO released from the nitroxy moiety of NCX-456 is responsible for caspase inhibition in this experimental setting (7, 29).

NO has emerged as a potent inhibitor of apoptosis in many cell types, and we and others (9, 15, 24-26) have previously demonstrated that a dominant mechanism for apoptosis prevention is the inhibition of caspase activity by nitrosation. NO-dependent caspase inhibition was demonstrated previously to prevent Bcl-2 cleavage and Cyt c release as well as activation of proteases that become activated after the release of Cyt c in hepatocytes and other cell types (24-27). Because of the capacity of NO to rapidly diffuse intracellularly and from cell to cell, this would represent an efficient mechanism to guard against the consequences of the activation of caspases, which might result from cell injury or exposure to other activators such as IFN-gamma , TNF-alpha , and FasL in the local environment (6, 7, 10, 13, 18, 21). Caspases are likely targets for nitrosation because their catalytic mechanisms require a redox-sensitive cysteine residue in the catalytic core (9). Previous studies carried out with purified subunits of caspases-1 and -3 demonstrated that the p17 subunit of caspase-3 and the p20 subunit of caspase-1 are selective targets for NO compounds and that the nitrosation of these subunits leads to a concentration-dependent inhibition of enzyme activity (9, 27, 28). More generally, there is now evidence that nitrosation is a mechanism that is extensively involved in caspase regulation (10, 19, 24-28). A recent report from Mannick et al. (27) indicates that in resting human cell lines caspase-3 zymogens are nitrosated and denitrosated on Fas/FasL cross-linking, indicating that caspase activation requires both denitrosation and zymogen cleavage. Although the reversal of NO-mediated inhibition by DTT is consistent with nitrosation being the main mechanism for caspase inhibition by NCX-456 and our results demonstrated NO formation in colon cell lines incubated with this compound, NO radical does not effectively nitrosate thiol groups, suggesting that a NO reaction product is implicated in caspase nitrosation (10, 17). NO+ equivalent nitrosates thiols and mimics the effect of NO donors when added to cells (10). Reactive nitrogen oxide species, including NO+ and its equivalents, can be generated by the reaction of NO with O2 or iron-sulfur clusters (17). These complexes have been shown to carry out transnitrosative reactions (10). In our in vitro experiments, the cells were grown under aerobic conditions and the interaction of NO with O2 could form N2O3, which has been shown to cause nitrosation via the formation of NO+ equivalents.

The failure of DTT treatment to induce a full recovery of all caspase-8 activity, however, raises the possibility that NO may also suppress caspase-8 activation (27). Supporting this concept, NCX-456 markedly reduced the amount of the caspase-8 p17 subunit released under Fas cross-linking (Fig. 6). Indeed, because caspase-8 activation is partially due to the autocatalytic cleavage of the inactive proenzyme, it cannot be excluded that S-nitrosation/inhibition of caspase-8 reduces the amount of active enzyme that is further generated through this pathway (19, 37). However, because caspase-8 may be activated via caspase-6 and NCX-456 behaves as a pancaspase inhibitor, it cannot be excluded that the NO-mesalamine derivative acts on multiple points of the caspase pathway (26).

In summary, we demonstrated that in contrast to mesalamine, NCX-456, an NO-mesalamine derivative, protects IFN-gamma -sensitized colon epithelial cells from death induced by TNF-alpha and Fas. The ability of NCX-456 to interfere with TNF-alpha -mediated signals may be of clinical relevance, considering that anti-TNF-alpha therapies effectively reduce inflammation in IBD patients. The ability of NCX-456 to prevent caspase activation may contribute to further downregulation of TNF-alpha signals in the inflamed mucosa and may hold some promise for biological therapies to attenuate T cell-dependent inflammation in IBD patients (11).


    ACKNOWLEDGEMENTS

We thank Barbara Federici for technical assistance.


    FOOTNOTES

This work was supported in part by a grant from Ministero dell' Universita' e della Ricerca Scientifica (MURST) to S. Fiorucci.

Address for reprint requests and other correspondence: S. Fiorucci, Clinica di Gastroenterologia ed Endoscopia Digestiva, Policlinico Monteluce, 06100 Perugia, Italy (E-mail: fiorucci{at}unipg.it).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10 November 2000; accepted in final form 30 April 2001.


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DISCUSSION
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