Crohn s disease is characterized by infiltration of activated

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1 ORIGINAL ARTICLE Remission-inducing Effect of Anti-TNF Monoclonal Antibody in TNBS Colitis: Mechanisms Beyond Neutralization? Chong Shen,* Gert de Hertogh, Dominique M.A. Bullens,* Gert Van Assche, Karel Geboes, Paul Rutgeerts, and Jan L. Ceuppens* Background: Tumor necrosis factor (TNF) plays an important role in the pathogenesis of several inflammatory diseases. Its expression is increased in inflamed mucosa of Crohn s disease patients and anti-tnf treatment improves mucosal inflammation. Besides neutralization, induction of apoptosis of monocytes/macrophages and T cells is thought to be an important mechanism of action of the anti-tnf monoclonal antibody therapy. The aim was to investigate the pathogenic role of TNF in hapten-induced colitis models and to study the relationship between apoptosis induction and disease remission. Methods: In 2 murine colitis models (trinitrobenzene sulphonic acid, TNBS, and oxazolone colitis), mice were injected daily with anti-tnf monoclonal antibody (mab). Macrophages were collected from lamina propria of TNBS colitis mice. 7AAD and anti-activecaspase-3 staining were used to study DNA degradation and intracellular caspase activation. A pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyketone (Z-VAD-FMK), was given to a subgroup of the colitis mice. Results: Treatment with anti-tnf effectively reduced intestinal mucosal inflammation in TNBS colitis but not in oxazolone colitis. Effectiveness was evidenced by a more rapid recovery of body weight and reduced cell infiltration, and downregulation of proinflammatory cytokines interferon-gamma (IFN- ), TNF, and IL-18 at the mrna level. Apoptosis was induced in lamina propria macrophages after treatment with anti-tnf, and it was abrogated through short-term pretreatment with Z-VAD-FMK. Conclusion: Anti-TNF downregulates proinflammatory cytokines and decreases cell infiltration in the bowel after TNBS application. The remission-inducing effect of anti-tnf may partly rely on apoptosis induction. Received for publication May 25, 2006; accepted September 12, From the *Laboratory of Experimental Immunology; Division of Gastroenterology; Division of Pathology, Katholike Universiteit Leuven, Belgium. Supported by grants from Schering-Plough, Centocor, and the FWO (Fund for Scientific Research) Vlaanderen (grant G ). Reprints: J.L. Ceuppens, Laboratory of Experimental Immunology, Campus Gasthuisberg (O&N), Herestraat 49, B-3000 Leuven, Belgium ( Jan.Ceuppens@uz.Kuleuven.be) Copyright 2006 Crohn s & Colitis Foundation of America, Inc. DOI /ibd Published online 19 December 2006 in Wiley InterScience (www. interscience.wiley.com). 308 (Inflamm Bowel Dis 2007;13: ) Key Words: TNBS, oxazolone, anti-tnf, caspase inhibitor, apoptosis Crohn s disease is characterized by infiltration of activated leukocytes in the intestinal mucosa with local production of proinflammatory cytokines 1 such as tumor necrosis factor (TNF), IL-12, IL-15, IL-18, and IFN TNF is a proinflammatory cytokine secreted predominantly by monocytes/macrophages but also by T cells, B cells, NK cells, and mast cells. Increased expression of TNF has been observed in the inflamed mucosa of inflammatory bowel disease 2 and in murine colitis models. 6 Recently, several groups showed that the key mechanism of action of anti-tnf monoclonal antibody (mab) infliximab might be the induction of apoptosis of activated monocytes and T lymphocytes in the inflamed gut. 7,8 In a previous study we have shown that infliximab induces apoptosis of monocytes and T cells both in vitro 9 and in vivo in a SCID-chimeric mouse model. 10 Infliximab-induced apoptosis in vitro was abrogated by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyketone (Z-VAD-FMK). 9 This compound is a cell-permeable pan-caspase inhibitor that irreversibly binds to the catalytic site of caspase proteases and inhibits apoptosis. 11,12 The data implied that apoptosis induction was caspase-dependent. In this study we evaluated the effect of a chimeric rat antimouse TNF mab in 2 models of colitis. TNBS colitis model is an IL-12 driven, Th1-mediated response. 13 TNF is necessary for both the initiation and persistence of the Th1 response, possibly by acting as a proximal cofactor for IL-12 or IL-18 production. 14 Elevated lesional TNF was found and anti-tnf was proved to attenuate the colitis. Furthermore, colitis could not be induced in TNF-deficient mice. 6 The nature of the inflammation at both the macroscopic and microscopic levels in TNBS models closely resembles Crohn s disease. We also studied the oxazolone (4-ethoxymethylene-2phenyl-2oxazoline-5-one)-induced colitis for comparison. The latter is a Th2 type model that is IL-4-driven and marked by elevated IL-4, IL-5, and IL-13 production and thought to be more similar to ulcerative colitis. 15,16 Whether there is also elevated TNF production is not clear. We further investigated whether anti-tnf induces apoptosis of mono- Inflamm Bowel Dis Volume 13, Number 3, March 2007

2 Inflamm Bowel Dis Volume 13, Number 3, March 2007 Remission-inducing Effect of Anti-TNF cytes/macrophages in the inflamed lamina propria in colitic mice. MATERIALS AND METHODS Mice C57 BL/6 male mice (4 5 weeks) were obtained from M&B (Denmark). Mice were bred under specific pathogenfree conditions and maintained in the Animal Care Facility of the Faculty of Medicine, Gasthuisberg, Catholic University of Leuven (Belgium). All mice were raised under specific pathogen-free conditions in microisolator cages with filtered air and were fed autoclaved food and water. Mice were 4 5 weeks of age when experiments started. Antibodies and Reagents The antibody to TNF used for in vivo treatment is a modified rat-mouse Fc chimeric antibody derived from an original rat antimouse TNF mab (cv1q, IgG2a), and together with its control antibody (cvam, IgG2a), were kindly provided by Dr. D. Shealy (Centocor, Malvern, PA). PE-labeled anti-mouse F4/80 (CI:A3-1, rat IgG2b, directed specifically against a murine macrophage-restricted cell surface glycoprotein), was purchased from Serotec (Oxford, UK). A fluorescein isothiocyanate (FITC)-conjugated monoclonal active caspase-3 antibody apoptosis kit was purchased from Becton Dickinson (Erembodegem, Belgium). Seven amino actinomycin-d (7AAD) was obtained from Novabiochem (La Jolla, CA). N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyketone (Z-VAD-FMK) was purchased from BD Biosciences (San Jose, CA). Establishment of Hapten (TNBS or Oxazolone)- Induced Colitis and Anti-TNF Treatment Colitis was induced by rectal administration challenge of 1 mg TNBS or oxazolone in 50% ethanol after 2 times cutaneous presensitization. Briefly, mice were sensitized twice 10 days and 5 days before challenge. For sensitization, a2 2 cm field of the abdominal skin was shaved and 100 L of 5 mg TNBS or oxazolone in 50% ethanol solution was applied. On the day of challenge, mice were first lightly anesthetized with metofane, subsequently TNBS or oxazolone was administered per rectum by a round-tip needle equipped with a 1-mL syringe. The tip of the needle was inserted so that the tip was cm proximal to the anal verge and TNBS or oxazolone was injected with a total volume of 100 L. To ensure distribution of hapten within the entire colon and cecum, mice were held in a vertical position for 1 minute after the injection. Subsequently, 100 g anti-tnf (cv1q) or its isotype control (cvam) was injected intraperitoneally (daily starting from day 0 until the end of the experiment). Histology Evaluations Both macroscopic and microscopic histology evaluations were performed as described previously. 17 The colon was excised, immediately examined visually, and damage was scored on a 0 12 scale. Colon sections were then fixed in 6% formalin and embedded in paraffin, cut into sections, and then stained with hematoxylin and eosin. Stained sections were examined for evidence of colitis using as criteria the presence of cell infiltration, elongation and/or distortion of crypts, crypt abscesses, reduction in goblet cell number, frank ulceration, and edema formation. Measurement of Cytokines; Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Colon from TNBS or oxazolone colitic mice was immediately frozen in liquid nitrogen after dissection and stored at 70 C until extraction of total RNA as described previously. 17 A constant amount of 1 g of total RNA was used for oligo-(dt)-primed cdna synthesis (Ready-to-go-kit, Pharmacia, Uppsala, Sweden). After 90 minutes at 37 C, the reverse transcriptase was inactivated by incubating the cdna samples for 5 minutes at 95 C. The amount of cdna was quantified by real-time RT-PCR using specific primers for -actin, TNF, IFN-, and IL-18, with the ABI Prism 7700 Sequence Detection System (SDS) from Applied Biosystems (Foster City, CA). PCR was performed in a total volume of 25 L, containing 5 L cdna and 20 L mix with the TaqMan Universal PCR Master Mix (2X) (Applied Biosystems) combined with 300 nm of the primer and 100 nm of the probe. They were performed in the following conditions: 10 minutes at 95 C followed by 40 cycles of 15 seconds at 95 C and 1 minute at 60 C. Levels of cytokine mrna expression are presented as a ratio after normalization to the housekeeping gene -actin. Measurement of MPO Activity MPO activity was measured using a method described previously. 17,18 In brief, 2 days after intracolonic injection of TNBS or oxazolone, 50 mg colon was removed, homogenized, and sonicated on ice. The samples were frozen in liquid nitrogen and subsequently water-bathed at 37 C, each step lasted 3 minutes, and was repeated for 2 cycles. After centrifugation, an aliquot of the supernatant was then allowed to react with a solution of tetra-methyl-benzidine (1.6 mm) and 0.1 mm H 2 O 2. The rate of change in absorbance was measured spectrophotometrically at 460 nm. MPO activity was defined as the quantity of enzyme degrading 1 mol of peroxide/min at 37 C and was expressed in units per gram weight of wet tissue. Sample enzyme activity was measured from a standard curve of known MPO unit activity (assay sensitivity units per well). 309

3 Shen et al Inflamm Bowel Dis Volume 13, Number 3, March 2007 hours mice were sacrificed and blood mononuclear cells and laminar propria mononuclear cells (LPMCs) were collected for apoptosis analysis. Determination of Apoptosis by 7AAD and Antiactive-caspase-3 Staining Lamina propria macrophages from colonic tissue of colitic TNBS mice were isolated as described previously. 4 7AAD was used to study apoptosis as previously described. 10 In brief, cells were first stained with antimouse F4/80 (anti- FIGURE 1. Effect of administration of anti-tnf on body weight loss in mice suffering from TNBS- or oxazolone-induced colitis. Colitis was induced at day 0 by intrarectal administration of TNBS or oxazolone as described in Materials and Methods. Anti- TNF was injected intraperitoneally daily starting at day 0 before disease induction until day 10. Control mice received isotype control in an identical schedule. The data shown represent average values SEM. Each group contained 12 mice and data are from 2 separate experiments. The 1-way analysis of variance (ANOVA) test was used to analyze whether the differences of weight among the various groups were significant. An unpaired t-test was used to identify differences at each timepoint between experimental treatments. (*P 0.05, **P 0.01). A: TNBS colitis (anti-tnf versus isotype control). B: Oxazolone colitis - (anti-tnf versus isotype control). Z-VAD-FMK Treatment In a separate experiment, the pan-caspase inhibitor Z- VAD-FMK (200 g) was injected intraperitoneally 1 hour before TNBS instillation in the rectum. TNBS colitis was induced as described previously. One hour later, anti-tnf mab or its isotype control antibody was injected. After 12 FIGURE 2. Disease scores for anti-tnf-treated or control mice with hapten-induced colitis. Mice were sacrificed on day 2 and colons were resected for analysis at day 2. Macroscopic (A) and microscopic (B) scores were quantified as described previously. 17 Values are shown as mean SEM. The results are from 8 mice in each group (*P 0.05). 310

4 Inflamm Bowel Dis Volume 13, Number 3, March 2007 Remission-inducing Effect of Anti-TNF FIGURE 3. Histologic pictures of TNBS-induced colitis. Mice were sacrificed and colons were resected for analysis at day 2 after TNBS application. A, B: TNBS colitis mice treated with control antibody. C, D: TNBS colitis mice treated with anti-tnf. Magnification was 10 in A and C and 40 in B and D. The figure is representative of 12 mice in each group. [Color figure can be viewed in the online issue, which is available at macrophage mab) for 30 minutes. After washing, 5 L of 7AAD solution was added to the cell suspension for 10 minutes at 4 C protected from light. Intracellular activecaspase-3 staining was performed according to the manufacturer s instructions. Data on 10,000 events were acquired and processed using Cellquest software. Statistical Analysis Statistical analysis was performed with Prism Mac 3.0. All analyses were performed as comparison of experimental groups with respective controls by Mann Whitney U-testing, or by Kruskal Wallis testing where appropriate. All tests were 2-sided and values of P 0.05 were considered significant. Data are presented as means SEM. RESULTS Anti-TNF Treatment Reverses TNBS But Not Oxazolone Colitis Administration of hapten (either TNBS or oxazolone) to presensitized mice resulted in a 15% 20% decrease in body weight after 1 (TNBS) to 3 days (oxazolone). In TNBS colitis, animals recovered to their original body weight 8 days after disease induction. Daily intraperitoneal administration of anti-tnf accelerated recovery (Fig. 1A). Already at day 3, animals that received anti-tnf had regained their original weight. However, the therapeutic effect of anti-tnf could not be seen in the oxazolone colitis model (Fig. 1B). Separate experiments were performed in which animals were sacrificed 2 days after colitis induction. Disease severity was scored macroscopically and histologically (Fig. 2). Anti- TNF significantly reduced disease scores in TNBS colitis, but not in oxazolone colitis. Representative histological pictures are shown in Figures 3 and 4. In oxazolone colitic mice or TNBS colitic mice treated with control antibody (cvam), the entire colonic wall was edematous and mucosal lesions were observed throughout the colon. The ulcers often penetrated the colon, resulting in adherence to adjacent tissues. Histologically, distortion of crypts, loss of goblet cells, and infiltration of mononuclear cells were observed in all mice. Some parts of the mucosal layer lost crypts and were replaced with lymphocytes, macrophages, and fibrotic tissue (Figs. 3, 4). Compared to the TNBS colitis, oxazolone colitis displays more marked edema in the submucosal layer. A superficial inflammation characterized by the presence of epithelial cell loss and patchy ulceration, pronounced depletion of mucin-producing goblet 311

5 Shen et al Inflamm Bowel Dis Volume 13, Number 3, March 2007 FIGURE 4. Histologic pictures of oxazolone-induced colitis. Mice were sacrificed and colons were resected for analysis at day 2 after oxazolone application. A, B: Oxazolone colitis mice treated with control antibody. C, D: Oxazolone colitis mice treated with anti-tnf. Magnification was 10 in A and C and 40 in B and D. The figure is representative of 12 mice in each group. [Color figure can be viewed in the online issue, which is available at cells, and reduction of the density of the tubular glands was present. All these features were clearly reduced after anti- TNF treatment in the TNBS model, but not in the oxazolone model. MPO Activity in Anti-TNF-Treated Colitic Mice Colon tissue from day 2 after disease induction was used for measuring MPO activity. Figure 5 illustrates a significant decrease of tissue MPO activity in anti-tnf treated TNBS colitic mice. MPO activity was reduced almost 2-fold ( U/g compared to U/g in control mice). The reduced tissue MPO activity suggests that anti-tnf reduced neutrophil and/or macrophage infiltration. These data correlate with the microscopic analysis, where less cell infiltration was observed. In contrast, anti-tnf did not have evident effect on MPO activity in oxazolone-induced colitis ( U/g in treated mice compared to U/g control treatment). Anti-TNF Treatment Downregulates Lesional TNF, IFN-, and IL-18 in TNBS Colitic Mice In order to address whether cytokine production by T cells and/or macrophages were affected after anti-tnf treat- FIGURE 5. MPO activity in colon tissue at day 2 after induction of colitis. Mice were sacrificed and colons were resected at day 2 after disease induction. MPO activity was defined as the quantity of enzyme degrading 1 mol peroxide/min at 37 C and expressed in units per gram weight of tissue. Mice were treated with either anti-tnf or its isotype control. Results represent mean SEM of 6 mice (*P 0.05). 312

6 Inflamm Bowel Dis Volume 13, Number 3, March 2007 Remission-inducing Effect of Anti-TNF ment, real-time RT-PCR was performed to qualify TNF, IFN-, and IL-18 mrna on colon homogenates obtained from animals sacrificed at day 2 after disease induction. TNF is clearly upregulated in TNBS colitis, but not in oxazolone colitis. Anti-TNF therapy significantly reduced TNF mrna. IFN- mrna was evidently reduced when anti-tnf was given in the TNBS model, but the difference did not reach statistical significance (Fig. 6). IL-18 is a macrophage-derived cytokine and a potent inducer of IFN- for intestinal lymphocytes. 19 Anti-IL-18 mab was shown to result in a dramatic attenuation of TNBS colitis. 19 IL-18 mrna in anti-tnf-treated TNBS colitis mice was also significantly reduced (Fig. 6), pointing toward decreased macrophage inflammation and/or activity. Surprisingly, none of these inflammatory cytokines was upregulated in oxazolone colitis. Anti-TNF Induces Apoptosis of LP Macrophages in TNBS Colitis Mice We next investigated a potential apoptosis-inducing effect of antimurine TNF in TNBS colitis. Colonic lamina propria mononuclear cells were collected from colitic mice that were either sham (isotype)-treated or had received anti- TNF treatment 12 hours before. Part of the mice was pretreated once with the pan-caspase inhibitor, Z-VAD-FMK. Figure 7 illustrates the induction of 7AAD reactivity (which reflects apoptosis) and the activation of caspase-3 intracellularly in the isolated laminar propria (LP) macrophages of TNBS colitis mice. As shown in Figure 8, the percentage of 7AAD-positive macrophages were significantly higher in anti-tnf-treated mice ( ) compared to that of control antibody-treated mice ( ). Z-VAD-FMK pretreatment abolished apoptosis induction by anti-tnf; only ( ) 7AAD-positive macrophages could be detected. Anti-active-caspase 3 staining shows similar results. After anti-tnf treatment a higher percentage ( ) of positive cells was found compared to mice treated with control antibody ( ). Caspase 3 activation was reduced but could not be completely inhibited by Z-VAD-FMK, as there were still % positive cells. DISCUSSION The present studies demonstrate that treatment with anti-tnf in TNBS colitis effectively reduced inflammatory cell infiltration and down-regulated lesional IL-18, TNF, and IFN- secretion. We also show here an elevated local TNF production in the TNBS colitis, and down-regulation of the production by anti-tnf. Increased expression of TNF in inflamed colon might be attributed mainly to mucosal macrophage activation after luminal antigen triggering, but might also be secreted by activated T cells. 20 These data on production and on the effect of anti-tnf provide further evidence that TNF is an important proinflammatory mediator in the induction of intestinal inflammation in this model of FIGURE 6. TNF, IFN-, and IL-18 mrna quantification in the colon of colitis mice. Mice were treated with anti-tnf or isotype control mab and sacrificed at day 2 after disease induction with TNBS or oxazolone. Colons were resected for analysis and TNF, IFN-, and IL-18 mrna were quantified by real-time RT-PCR. mrna expression is presented as a ratio after normalization to the housekeeping gene -actin and multiplied by 10 6 for IFN- and TNF, and 10 4 for IL-18. Results are expressed as mean SEM for 6 mice (*P 0.05; n.s., no significant difference). 313

7 Shen et al Inflamm Bowel Dis Volume 13, Number 3, March 2007 FIGURE 7. Anti-TNF induces apoptosis of lamina propria macrophages from TNBS colitis mice. Z-VAD-FMK or vehicle (phosphatebuffered saline, PBS) was injected intraperitoneally and TNBS colitis was induced by intrarectal challenge as described previously. One hour later, anti-tnf mab or received isotype control IgG was given intraperitoneally. Mice were sacrificed 12 hours later and lamina propria mononuclear cells were isolated as described in Materials and Methods. The figure is representative of 8 mice in each group from 3 independent experiments. Numbers shown in each figure from the gated area (M1) represent the percentages of 7AAD( ) (left panel) or active-caspase 3( ) cells (right panel). (I) control antibody PBS (II) anti-tnf PBS (III) control antibody Z- VAD-FMK (IV) anti-tnf Z-VAD-FMK. [Color figure can be viewed in the online issue, which is available at com.] colitis. Oxazolone was reported to be a Th2 model with elevated IL-4 and IL ,16 In our study, neither IFN- nor TNF was significantly upregulated in oxazolone colitis mice. Therefore, it is not surprising to find the ineffectiveness of anti-tnf treatment of oxazolone colitis. The data suggest that TNF has no or a limited role in this model. Several potential mechanisms by which anti-tnf treatment exerts its beneficial effect have been proposed. There is increasing evidence that apoptosis represents an important event during gut inflammatory responses, allowing strict control of clonal expansion of immune cells in response to a broad spectrum of antigenic stimuli, as well as in the clear- 314

8 Inflamm Bowel Dis Volume 13, Number 3, March 2007 Remission-inducing Effect of Anti-TNF FIGURE 8. Anti-TNF induces apoptosis of lamina propria macrophages from TNBS colitis mice. The experimental protocol and FACS setting are described in the legend of Figure 7. This figure summarizes the findings for 8 mice in each group. A: Percent 7AAD ( ) cells among lamina propria macrophages. B: Percent caspase 3 ( ) cells among lamina propria macrophages. The groups are as in Figure 7. (I) control antibody PBS (II) anti- TNF PBS (III) control antibody Z-VAD-FMK (IV) anti-tnf Z- VAD-FMK (*P 0.05, ***P 0.001, n.s., no significant differences). ance of invading pathogenic organisms. Several apoptosisrelated signaling and intercellular mechanisms in Crohn s disease have been studied such as Bcl-2/Bax imbalance, Fasl-Fas, and caspase activation. One of the proposed mechanisms for the anti-tnf effect is induction of inflammatory cell apoptosis. We could indeed show here that anti-tnf induces apoptosis of monocytes/macrophages in vivo in colitic mouse. Elevated numbers of apoptotic monocytes/macrophages were observed in the LP in anti-tnf-treated TNBS colitis mice. This is further confirmed by staining for activecaspase 3. Procaspase-3, as all caspases and most proteases, is synthesized as an inactive proenzyme. 21 It is processed as active caspase-3 by an upstream caspase, which underlies initiator (not effector) procaspase activation. Also, reversal of apoptosis induction by treatment with the pan-caspase inhibitor Z-VAD-FMK points to apoptosis as the underlying mechanism for cell death. However, whether the remission-inducing effect of anti-tnf is caused by apoptosis-induction of inflammatory cells is still not clear. Apoptosis induction might be a late and indirect effect anti-tnf treatment, resulting from reduced presence of inflammation-induced survival factors such as IL-6. The latter cytokine indeed has been shown to contribute to the resistance of inflammatory cells in IBD to apoptosis induction. 22 In a recent study by Mitoma et al, 23 infliximab binding (but not etanercept) induced multiple antiinflammatory responses: for instance, enhanced G0/G1 cell cycle arrest, upregulated E-selectin expression, and IL-10 production; it also resulted in the activation of JNK/p53 and the upregulation of proapoptotic molecules like Bax and Bak. Marini et al 24 showed in SAMP/Yitc ileitis mice that TNF neutralization led to reduced Fas/CD95 expression of epithelial cells and resulted in amelioration of chronic inflammation. Similar findings were also reported by Diebel et al. 25 In conclusion, the present findings, together with other reports, thus prove that immune target therapy directed against TNF effectively blocks immunopathology in the mouse model of TNBS-induced colitis. Downregulation of proinflammatory cytokine secretion and of cell infiltration was clearly shown. However, TNF is not essential in all colitis models, as oxazolone colitis was not affected by anti- TNF treatment. Although we believe that anti-tnf-induced apoptosis of inflammatory cells contributes to the beneficial effects of infliximab in Crohn s disease, further understanding of the mechanisms involved in the events downstream to the binding to tmtnf is needed. REFERENCES 1. Fiocchi C. Inflammatory bowel disease: etiology and pathogenesis. Gastroenterology. 1998;115: Braegger CP, MacDonald TT. Immune mechanisms in chronic inflammatory bowel disease. Ann Allergy. 1994;72: Monteleone G, Biancone L, Marasco R, et al. Interleukin 12 is expressed and actively released by Crohn s disease intestinal lamina propria mononuclear cells. Gastroenterology. 1997;112: Liu Z, Geboes K, Colpaert S, et al. IL-15 is highly expressed in inflammatory bowel disease and regulates local T cell-dependent cytokine production. J Immunol. 2000;164: Monteleone G, Trapasso F, Parrello T, et al. Bioactive IL-18 expression is up-regulated in Crohn s disease. J Immunol. 1999;163: Neurath MF, Fuss I, Pasparakis M, et al. Predominant pathogenic role of tumor necrosis factor in experimental colitis in mice. Eur J Immunol. 1997;27:

9 Shen et al Inflamm Bowel Dis Volume 13, Number 3, March Lugering A, Schmidt M, Lugering N, et al. Infliximab induces apoptosis in monocytes from patients with chronic active Crohn s disease by using a caspase-dependent pathway. Gastroenterology. 2001;121: Van den Brande JM, Braat H, van den Brink GR, et al. Infliximab but not etanercept induces apoptosis in lamina propria T-lymphocytes from patients with Crohn s disease. Gastroenterology. 2003;124: Shen C, Van Assche G, Colpaert S, et al. Adalimumab induces apoptosis of human monocytes: a comparative study with infliximab and etanercept. Alimentary Pharmacology and Therapeutics,. 2005;21: Shen C, Maerten P, Van Assche G, et al. Infliximab induces apoptosis of monocytes and T lymphocytes in a human-mouse chimeric model. Clini Immunol. 2005;115: Gregoli PA, Bondurant MC. Function of caspases in regulating apoptosis caused by erythropoietin deprivation in erythroid progenitors. J Cell Physiol. 1999;178: Schrantz N, Blanchard DA, Auffredou MT, et al. Role of caspases and possible involvement of retinoblastoma protein during TGFbeta-mediated apoptosis of human B lymphocytes. Oncogene. 1999;18: Morris GP, Beck PL, Herridge MS, et al. Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology. 1989;96: Strober W, Fuss IJ, Blumberg RS. The immunology of mucosal models of inflammation. Annu Rev Immunol. 2002;20: Boirivant M, Fuss IJ, Chu A, et al. Oxazolone colitis: a murine model of T helper cell type 2 colitis treatable with antibodies to interleukin 4. J Exp Med. 1998;188: Heller F, Fuss IJ, Nieuwenhuis EE, et al. Oxazolone colitis, a Th2 colitis model resembling ulcerative colitis, is mediated by IL-13-producing NK-T cells. Immunity. 2002;17: Shen C, Bullens D, Kasran A, et al. Inhibition of glycolipid biosynthesis by N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin protects against the inflammatory response in hapten-induced colitis. Int Immunopharmacol. 2004;4: Mullane KM, Kraemer R, Smith B. Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischemic myocardium. J Pharmacol Methods. 1985;14: Maerten P, Shen C, Colpaert S, et al. Involvement of interleukin 18 in Crohn s disease: evidence from in vitro analysis of human gut inflammatory cells and from experimental colitis models. Clin Exp Immunol. 2004;135: Sands BE. Why do anti-tumor necrosis factor antibodies work in Crohn s disease?. Rev Gastroenterol Disord. 2004;4(Suppl 3):S Thornberry NA, Lazebnic Y. Caspase: enemies within. Science. 1998; 281: Atreya R, Mudter J, Finotto S, et al. Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in Crohn disease and experimental colitis in vivo. Nat Med. 2000;5: Mitoma H, Horiuchi T, Hatta N, et al. Infliximab induces potent antiinflammatory responses by outside-to-inside signals through transmembrane TNF-alpha. Gastroenterology. 2005;128: Marini M, Bamias G, Rivera-Nieves J, et al. TNF-alpha neutralization ameliorates the severity of murine Crohn s-like ileitis by abrogation of intestinal epithelial cell apoptosis. Proc Natl Acad Sci U S A.2003;100: Diebel LN, Liberati DM, Baylor AE 3rd, et al. The pivotal role of tumor necrosis factor-alpha in signaling apoptosis in intestinal epithelial cells under shock conditions. J Trauma. 2005;58: