Different sensitivity of lamina propria T-cell subsets to nitric oxide-induced apoptosis explains immunomodulatory activity of a nitric oxide-releasing derivative of mesalamine in rodent colitis

BACKGROUND & AIMS: Uncontrolled T-cell activation plays a critical role in the pathogenesis of inflammatory bowel diseases. Therefore, pharmacological strategies directed toward restoring the normal responsiveness of the immune system could be effective in the treatment of these pathologic conditions. The addition of a nitric oxide-releasing moiety to conventional drugs, such as aspirin and other anti-inflammatory analgesic drugs, results in new chemical entities with potent immunomodulatory activities. The aim of this study was to investigate the immunomodulatory activity of a nitric oxide-releasing derivative of mesalamine (NCX-456), as compared with standard mesalamine, in 2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice. METHODS: Cells and tissues from mice with 2,4,6-trinitrobenzene sulfonic acid-induced colitis and from interleukin 10-deficient mice with spontaneous chronic colitis receiving treatment with several doses of NCX-456 or mesalamine were analyzed for morphology, cytokine production, and apoptosis. RESULTS: NCX-456, but not mesalamine, administration resulted in a marked reduction in clinical, histological, and immunologic signs of colitis in both models. NCX-456 inhibited the release of T-helper type 1-derived cytokines and increased the release of the regulatory T cell-derived cytokines interleukin 10 and transforming growth factor beta. In vitro analyses showed that NCX-456 inhibited proliferation and caused selective apoptosis of the subset of activated lamina propria T-helper type 1 cells, whereas it was ineffective for regulatory T-cell function and survival. CONCLUSIONS: Collectively, these data show that NCX-456 inhibits lamina propria T-helper type 1 function and stimulates the activity of interleukin 10- and transforming growth factor beta-secreting cells, thus restoring mucosal immune homeostasis and suppressing intestinal inflammation.     

Remission-inducing effect of anti-TNF monoclonal antibody in TNBS colitis: mechanisms beyond neutralization?

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-active-caspase-3 staining were used to study DNA degradation and intracellular caspase activation. A pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (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-gamma), 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.     

Anti-interleukin 12 treatment regulates apoptosis of Th1 T cells in experimental colitis in mice.

BACKGROUND & AIMS: Trinitrobenzene sulfonic acid (TNBS)-induced colitis is a T-helper 1 (Th1) T cell-mediated inflammation that is rapidly reversed by administration of anti-interleukin (IL) 12. This study sought to define the mechanism of this curative effect. METHODS: Cells and tissue from mice with TNBS colitis receiving treatment with anticytokines were analyzed for phenotype, cytokine production, and apoptosis. RESULTS: In initial studies, we found that treatment of mice with TNBS-induced colitis with anti-IL-12 was more effective than with anti-interferon (IFN)-gamma, and that anti-IL-12 led to complete normalization of IFN-gamma production by lamina propria T cells ex vivo, whereas anti-IFN-gamma did not. These data suggesting that anti-IL-12 leads to reversal of colitis by elimination of the Th1 T cells were substantiated by studies showing that anti-IL-12 treatment led to increased numbers of apoptotic cells in the lamina propria and spleen by both TUNEL staining of tissues and dispersed spleen cell populations. Finally, we found that the observed apoptosis was mediated by the Fas pathway because (1) MRL/MpJ-lpr(fas) mice lacking Fas function develop colitis that responds poorly to treatment with anti-IL-12; and (2) SJL/J mice with TNBS colitis that received Fas-Fc to block the Fas pathway were resistant to anti-IL-12 treatment. CONCLUSIONS: These studies show that a main mechanism of action of anti-IL-12 in TNBS-induced colitis is the induction of Fas-mediated apoptosis of the Th1 T cells, causing inflammation.     

The in vitro anti-inflammatory effects of recombinant anti-CD25 immunotoxin on lamina propria T cells of patients with inflammatory bowel disease are not sufficient to cure experimental colitis in mice

BACKGROUND AND AIMS: In chronic inflammatory bowel disease (IBD) such as Crohn's disease and ulcerative colitis an aberrant mucosal immune regulation is observed accompanied by upregulation of proinflammatory cytokines. Lamina propria T cells of inflamed mucosa have an activated phenotype characterized by increased expression of surface markers such as CD25. We therefore determined the anti-inflammatory effect of a recombinant immunotoxin consisting of an anti-CD25 single chain variable fragment (scFv) fused to a deletion mutant of Pseudomonas exotoxin A [RFT5(scFv)ETA'] on isolated lamina propria lymphocytes of patients with IBD and in the murine model of trinitrobenzene sulfonic acid (TNBS) induced colitis. PATIENTS AND/METHODS: Lamina propria lymphocytes of 25 patients with IBD and 19 control patients were stimulated in absence or presence of RFT5(scFv)ETA'. Interferon-gamma production was determined in the supernatant by ELISA and the induction of apoptosis by flow cytometry after propidium iodide staining. BALB/c mice received TNBS intrarectally and were treated with RFT5(scFv)ETA'. RESULTS: In vitro the administration of RFT5(scFv)ETA' significantly reduced interferon-gamma production and increased apoptosis in lamina propria lymphocytes isolated of inflamed mucosa. However, this contrainflammatory regulation did not result in gain of weight or increased life span in experimental colitis in vivo. CONCLUSION: In addition to the downregulation of the proinflammatory cytokine in vitro, RFT5(scFv)ETA' induced neither a direct nor a bystander effect in an in vivo model of colitis. Therefore our data do not support potential therapeutic implications of targeting CD25 by RFT5(scFv)ETA' in chronic IBD.     

Inhibition of indoleamine 2,3-dioxygenase augments trinitrobenzene sulfonic acid colitis in mice

BACKGROUND & AIMS: Indoleamine 2,3-dioxygenase (IDO), an interferon gamma-induced intracellular enzyme, inhibits lymphocyte proliferation through tryptophan degradation. IDO is highly expressed in the mammalian intestine. We sought to determine whether IDO played a regulatory role in the T-cell helper 1 (Th1)-mediated trinitrobenzene sulfonic acid (TNBS) model of colitis. METHODS: Intrarectal TNBS was given to SJL/J mice along with either placebo or a specific IDO inhibitor. IDO protein and mRNA expression were assessed by Western blotting and real-time PCR. Colonic lamina propria mononuclear cells (LPMNCs) were isolated, fractionated, and cultured, in the presence and absence of IFN-gamma, to determine the cell type(s) expressing IDO. RESULTS: IDO is expressed by professional antigen-presenting cells in the lamina propria. Induction of TNBS colitis resulted in a significant increase in IDO mRNA (P = 0.005) and protein expression. IDO inhibition during TNBS colitis resulted in an 80% mortality compared with 10% for placebo-treated animals (P = 0.0089). IDO inhibition resulted in a more severe colitis both histologically and morphologically (P < 0.05) and significantly increased colonic proinflammatory cytokine expression compared with placebo-treated animals. CONCLUSIONS: IDO is expressed in the normal colon and is up-regulated in the setting of TNBS colitis. Inhibition of IDO during TNBS colitis resulted in increased mortality and an augmentation of the normal inflammatory response. These findings suggest that IDO plays an important role in the down-regulation of Th1 responses within the gastrointestinal tract.     

A beta-oxidation-resistant lipoxin A4 analog treats hapten-induced colitis by attenuating inflammation and immune dysfunction

Lipoxins and aspirin-triggered 15-epi-lipoxins (ATL) are counter-regulatory eicosanoids with potent antiinflammatory actions. Oral efficacy and mechanism of action of ZK-192, a beta-oxidation-resistant 3-oxa-ATL analog, were examined in trinitrobenzenesulphonate (TNBS)-induced colitis. When dosed orally once daily, 300 and 1,000 mug/kg ZK-192 markedly attenuated TNBS colitis in rodents both in preventive and therapeutic regimens. ZK-192 attenuated weight loss, macroscopic and histologic colon injury, mucosal neutrophil infiltration, and colon wall thickening. ZK-192 was as effective as 3-10 mg/kg oral prednisolone. ZK-192 decreased mucosal mRNA levels for several inflammatory mediators: inducible nitric oxide synthase, cyclooxygenase 2, and macrophage inflammatory protein 2. ZK-192 also decreased mucosal mRNA and protein levels of T helper 1 effector cytokines: tumor necrosis factor alpha, IL-2, and IFN-gamma. Systemic levels of these cytokines were also dramatically attenuated. CD3/CD28-mediated costimulation of T helper 1 effector cytokine release in lamina propria mononuclear cells was markedly inhibited by ZK-192 ex vivo and in vitro. ZK-192 also prevented colitis in lymphocyte-deficient severe combined immunodeficient mice, with approximately 75% inhibition of mucosal tumor necrosis factor alpha and IL-2 levels. The results are further evidence that innate immune cells function as triggers for hapten-induced colitis. The combined antiinflammatory and immunomodulatory effects of ZK-192 in TNBS colitis suggest that ATL analogs may be an attractive oral treatment approach for inflammatory bowel diseases.     

Inhibition of Smad7 with a specific antisense oligonucleotide facilitates TGF-beta1-mediated suppression of colitis

BACKGROUND & AIMS: Defective transforming growth factor (TGF)-beta1 signaling due to high levels of Smad7 is a feature of inflammatory bowel disease (IBD). In this study, we analyzed the effect of reducing Smad7 levels with antisense oligonucleotide on mouse models of colitis. METHODS: Mucosal samples taken from colitic tissue of mice with colitis due to either haptenating reagents (trinitrobenzene sulfonic acid [TNBS] or oxazolone) or to transfer of T cells (SCID transfer colitis) were analyzed for Smad3 and/or Smad7 expression by Western blotting and, in some cases, content of TGF-beta1 by enzyme-linked immunosorbent assay. The effect of oral Smad7 antisense oligonucleotide on mucosal inflammation was assessed. RESULTS: TGF-beta1 levels were increased in the inflamed tissues of mice with colitis induced by either TNBS or oxazolone. Nevertheless, TGF-beta1 did not exert a regulatory effect, probably because TGF-beta1 signaling was blocked, as indicated by the presence of reduced Smad3 phosphorylation and high levels of Smad7. Oral administration of Smad7 antisense oligonucleotide to colitic mice restored TGF-beta1 signaling via Smad3 and ameliorated inflammation in hapten-induced colitis. In addition, Smad7 antisense oligonucleotide had a therapeutic effect on relapsing TNBS-induced colitis but not on cell-transfer colitis. CONCLUSIONS: These data suggest that colitis models associated with high endogenous TGF-beta1 levels and defective TGF-beta1 signaling due to high levels of Smad7 can be ameliorated by down-regulation of Smad7 and by oral administration of Smad7 antisense oligonucleotide. This may represent a new approach to the control of IBD, particularly during active phases when its Smad7 profile resembles that of hapten-induced colitis.     

Monoclonal anti-interleukin 23 reverses active colitis in a T cell-mediated model in mice

BACKGROUND & AIMS: Interleukin (IL)-23 supports a distinct lineage of T cells producing IL-17 (Th17) that can mediate chronic inflammation. This study was performed to define the role of IL-23 and Th17 cells in chronic colitis in mice. METHODS: Colitis was induced by transfer of a cecal bacterial antigen-specific C3H/HeJBir (C3Bir) CD4(+) T-cell line to C3H/HeSnJ SCID mice. Cytokines were measured by flow cytometry, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction. Monoclonal anti-IL-23p19 was administered at the same time as or 4 weeks after pathogenic CD4 T-cell transfer. A histopathology colitis score was assessed in a blinded fashion. RESULTS: The pathogenic C3Bir CD4(+) T-cell line contained more cells producing IL-17 than those producing interferon-gamma and these were distinct subsets; after adoptive transfer to SCID recipients, Th17 cells were predominant in the lamina propria of mice with colitis. Bacteria-reactive CD4(+) Th1 and Th17 lines were generated. The Th17 cells induced marked inflammation in a dose-dependent manner. Even at a dose as low as 10(4) cells/mouse, Th17 cells induced more severe disease than Th1 cells did at 10(6) cells/mouse. Monoclonal anti-IL-23p19 prevented and treated active colitis, with down-regulation of a broad array of inflammatory cytokines and chemokines in the colon. Anti-IL-23p19 induced apoptosis in colitogenic Th17 cells in vitro and in vivo. CONCLUSIONS: Bacterial-reactive CD4(+) Th17 cells are potent effector cells in chronic colitis. Inhibition of IL-23p19 was effective in both prevention and treatment of active colitis. IL-23 is an attractive therapeutic target for inflammatory bowel disease.