IL-6 is required for the development of Th1 cell-mediated murine colitis

Proinflammatory cytokines have been demonstrated to play a crucial role in the pathogenesis of Crohn's disease. Among those cytokines, strong expression of IL-6 has been repeatedly demonstrated. To examine the role for IL-6 in the pathogenesis of Crohn's disease, we introduced anti-IL-6R mAb to a murine model of colitis. Colitis was induced in C.B-17-scid mice transferred with CD45RBhigh CD4+ T cells from BALB/c mice. Anti-IL-6R mAb or rat IgG was administered weekly after T cell transfer. ICAM-1 and VCAM-1 expression were analyzed by immunohistochemistry. Colonic cytokine expression was determined by RT-PCR. Mice treated with mAb showed normal growth, whereas controls lost weight. The average colitis score was 0.64 for mAb-treated mice and 1.80 for controls. T cell expansion in treated mice was less remarkable than in the controls. Colonic ICAM-1 and VCAM-1 expression were markedly suppressed by mAb. IFN-gamma, TNF-alpha, and IL-1beta mRNA were reduced by the treatment. The results presented here show a crucial role for IL-6 in the pathogenesis of murine colitis and suggest a therapeutic potential of anti-IL-6R mAb for treatment of human Crohn's disease.     

Therapeutic effect of anti-OX40L and anti-TNF-alpha MAbs in a murine model of chronic colitis

Interaction of OX40 (CD134) on T cells with its ligand (OX40L) on antigen-presenting cells has been implicated in pathogenic T cell activation. This study was performed to explore the involvement of OX40/OX40L in the development of T cell-mediated chronic colitis. We evaluated both the preventive and therapeutic effects of neutralizing anti-OX40L MAb on the development of chronic colitis in SCID mice induced by adoptive transfer of CD4(+)CD45RB(high) T cells as an animal model of Crohn's disease. We also assessed the combination of anti-OX40L and anti-TNF-alpha MAbs to improve the therapeutic effect. Administration of anti-OX40L MAb markedly ameliorated the clinical and histopathological disease in preventive and therapeutic protocols. In vivo treatment with anti-OX40L MAb decreased CD4(+) T cell infiltration in the colon and suppressed IFN-gamma, IL-2, and TNF-alpha production by lamina propria CD4(+) T cells. The combination with anti-TNF-alpha MAb further improved the therapeutic effect by abolishing IFN-gamma, IL-2, and TNF-alpha production by lamina propria CD4(+) T cells. Our present results suggested a pivotal role of OX40/OX40L in the pathogenesis of T cell-mediated chronic colitis. The OX40L blockade, especially in combination with the TNF-alpha blockade, may be a promising strategy for therapeutic intervention of Crohn's disease.     

Blockade of B7-H1 suppresses the development of chronic intestinal inflammation

A newly identified costimulatory molecule, programmed death-1 (PD-1), provides a negative signal that is essential for immune homeostasis. However, it has been suggested that its ligands, B7-H1 (PD-L1) and B7-dendritic cells (B7-DC; PD-L2), could also costimulate T cell proliferation and cytokine secretion. Here we demonstrate the involvement of PD-1/B7-H1 and B7-DC interaction in the development of colitis. We first examined the expression profiles of PD-1 and its ligands in both human inflammatory bowel disease and a murine chronic colitis model induced by adoptive transfer of CD4(+)CD45RB(high) T cells to SCID mice. Second, we assessed the therapeutic potential of neutralizing anti-B7-H1 and/or B7-DC mAbs using this colitis model. We found significantly increased expression of PD-1 on T cells and of B7-H1 on T, B, and macrophage/DCs in inflamed colon from both inflammatory bowel disease patients and colitic mice. Unexpectedly, the administration of anti-B7-H1, but not anti-B7-DC, mAb after transfer of CD4(+)CD45RB(high) T cells suppressed wasting disease with colitis, abrogated leukocyte infiltration, and reduced the production of IFN-gamma, IL-2, and TNF-alpha, but not IL-4 or IL-10, by lamina propria CD4(+) T cells. These data suggest that the interaction of PD-1/B7-H1, but not PD-1/B7-DC, might be involved in intestinal mucosal inflammation and also show a possible role of interaction between B7-H1 and an as yet unidentified receptor for B7-H1 in inducing T cell activation.     

Dominant role for TL1A/DR3 pathway in IL-12 plus IL-18-induced IFN-gamma production by peripheral blood and mucosal CCR9+ T lymphocytes

The TNF-like cytokine TL1A augments IFN-gamma production by anti-CD3 plus anti-CD28 and IL-12/IL-18-stimulated peripheral blood (PB) T cells. However, only a small subset of PB T cells respond to TL1A stimulation with IFN-gamma production. PB CCR9+ T cells represent a small subset of circulating T cells with mucosal T cell characteristics and a Th1/Tr1 cytokine profile. In the current study, we show that TL1A enhanced IFN-gamma production by TCR- or CD2/CD28-stimulated CCR9(+)CD4+ PB T cells. However, TL1A had the most pronounced effect on augmenting IFN-gamma production by IL-12/IL-18-primed CCR9(+)CD4+ PB T cells. TL1A enhanced both the percentage and the mean fluorescence intensity of IFN-gamma in CCR9(+)CD4+ T cells as assessed by intracellular cytokine staining. IL-12 plus IL-18 up-regulated DR3 expression in CCR9(+)CD4+ T cells but had negligible effect on CCR9(-)CD4+ T cells. CCR9(+)CD4+ T cells isolated from the small intestine showed a 37- to 105-fold enhancement of IFN-gamma production when TL1A was added to the IL-12/IL18 cytokine combination. Cell membrane-expressed TL1A was preferentially expressed in CCR9(+)CD4+ PB T cells, and a blocking anti-TL1A mAb inhibited IFN-gamma production by cytokine-primed CCR9(+)CD4+ T cells by approximately 50%. Our data show that the TL1A/DR3 pathway plays a dominant role in the ultimate level of cytokine-induced IFN-gamma production by CCR9+ mucosal and gut-homing PB T cells and could play an important role in Th1-mediated intestinal diseases, such as Crohn's disease, where increased expression of IL-12, IL-18, TL1A, and DR3 converge in the inflamed intestinal mucosa.     

IL-15-dependent activation-induced cell death-resistant Th1 type CD8 alpha beta+NK1.1+ T cells for the development of small intestinal inflammation

To clarify the role of IL-15 at local sites, we engineered a transgenic (Tg) mouse (T3(b)-IL-15 Tg) to overexpress human IL-15 preferentially in intestinal epithelial cells by the use of T3(b)-promoter. Although IL-15 was expressed in the entire small intestine (SI) and large intestines of the Tg mice, localized inflammation developed in the proximal SI only. Histopathologic study revealed reduced villus length, marked infiltration of lymphocytes, and vacuolar degeneration of the villus epithelium, beginning at approximately 3-4 mo of age. The numbers of CD8(+) T cells, especially CD8alphabeta(+) T cells expressing NK1.1, were dramatically increased in the lamina propria of the involved SI. The severity of inflammation corresponded to increased numbers of CD8alphabeta(+)NK1.1(+) T cells and levels of production of the Th1-type cytokines IFN-gamma and TNF-alpha. Locally overexpressed IL-15 was accompanied by increased resistance of CD8alphabeta(+) NK1.1(+) T cells to activation-induced cell death. Our results suggest that chronic inflammation in the SI in this murine model is mediated by dysregulation of epithelial cell-derived IL-15. The model may contribute to understanding the role of CD8(+) T cells in human Crohn's disease involving the SI.     

Bacterial-reactive T regulatory cells inhibit pathogenic immune responses to the enteric flora

We showed previously that cecal bacterial Ag (CBA)-specific CD4(+) T cells induce colitis when transferred into SCID mice. The purpose of this study was to generate and characterize CBA-specific regulatory T cells in C3H/HeJBir (Bir) mice. CD4(+) T cells were stimulated with CBA-pulsed APC in the presence of IL-10 every 10-14 days. After four or more cycles, these T cells produced high levels of IL-10, low levels of IL-4 and IFN-gamma, and no IL-2, consistent with the phenotype of T regulatory-1 (Tr1) cells. Bir Tr1 cells proliferated poorly, but their proliferation was dependent on CD28-B7 interactions and was MHC class II-restricted. Transfer of Bir Tr1 cells into SCID mice did not result in colitis, and cotransfer of Bir Tr1 T cells with pathogenic Bir CD4(+) Th1 cells prevented colitis. Bir Tr1 cells inhibited proliferation and IFN-gamma production of a CBA-specific Th1 cell line in vitro. Such inhibition was partly due to IL-10 and TGFbeta1, but cognate interactions with either APCs or Th1 cells were also involved. Normal intestinal lamina propria CD4(+) T cells had Tr1-like activity when stimulated with CBA-pulsed APCs. We conclude that CD4(+) T cells with the properties of Tr1 cells are present in the intestinal lamina propria and hypothesize that these cells maintain intestinal immune homeostasis to the enteric flora.     

Characterization of Foxp3+CD4+CD25+ and IL-10-secreting CD4+CD25+ T cells during cure of colitis

CD4+CD25+ regulatory T cells can prevent and resolve intestinal inflammation in the murine T cell transfer model of colitis. Using Foxp3 as a marker of regulatory T cell activity, we now provide a comprehensive analysis of the in vivo distribution of Foxp3+CD4+CD25+ cells in wild-type mice, and during cure of experimental colitis. In both cases, Foxp3+CD4+CD25+ cells were found to accumulate in the colon and secondary lymphoid organs. Importantly, Foxp3+ cells were present at increased density in colon samples from patients with ulcerative colitis or Crohn's disease, suggesting similarities in the behavior of murine and human regulatory cells under inflammatory conditions. Cure of murine colitis was dependent on the presence of IL-10, and IL-10-producing CD4+CD25+ T cells were enriched within the colon during cure of colitis and also under steady state conditions. Our data indicate that although CD4+CD25+ T cells expressing Foxp3 are present within both lymphoid organs and the colon, subsets of IL-10-producing CD4+CD25+ T cells are present mainly within the intestinal lamina propria suggesting compartmentalization of the regulatory T cell response at effector sites.     

Trichuris muris: host intestinal epithelial cell hyperproliferation during chronic infection is regulated by interferon-gamma.

Chronic infection with the intestinal nematode Trichuris muris is associated with an inappropriate type 1 cytokine response (production of predominantly IFN-gamma), whereas resistance to infection requires the induction of a protective type 2 response with the production of interleukin (IL)-4, IL-5, IL-9, and IL-13. T. muris inhabits an intracellular niche within murine intestinal epithelial cells of the caecum and in common with other intestinal helminth infections is associated with gross morphological changes in gut architecture. The purpose of this study was to characterise cytokine production during chronic infection in AKR and severe-combined-immunodeficient (SCID) mice and investigate what effect the anti-parasite response had on epithelial cell proliferation and so regulation of intestinal pathology. Pulse labeling with tritiated thymidine is employed to generate a sensitive cell position-linked proliferation index of the intestinal epithelium at various times postinfection. Infection in AKR mice is characterized by a marked elevation in antigen specific IFN-gamma production from restimulated mesenteric lymph node cells and a significant increase in proliferation of pluripotent epithelial stem cells and transit cells within the crypts. Similarly, elevated IFN-gamma production was observed in the mesenteric lymph nodes and intestinal mucosa of infected SCID mice, with epithelial cell hyperproliferation and the development of crypt hyperplasia in the caecum. Critically, in vivo depletion of IFN-gamma during infection in SCID mice resulted in no significant increase in epithelial cell proliferation and effectively precluded the development of crypt hyperplasia without altering infection outcome. Taken together, the data provides the first detailed cell position linked analysis of epithelial dysregulation during chronic T. muris infection and identifies a critical role for IFN-gamma, either directly or indirectly, in regulation of epithelial cell proliferation during the chronic intestinal inflammation associated with infection.     

Neutralization of interleukin-18 reduces severity in murine colitis and intestinal IFN-gamma and TNF-alpha production

Interleukin (IL)-18, initially described as interferon (IFN)-gamma-inducing factor, is expressed in the inflamed mucosa of patients with Crohn's disease. To investigate the role of IL-18 in intestinal inflammation, the effect of neutralizing antimurine IL-18 antiserum in dextran sulfate sodium (DSS)-induced colitis in BALB/c and C57BL/6 mice was examined. During a dose response of DSS, levels of colonic IL-18 increased parallel with clinical worsening. With the use of confocal laser microscopy, the increased IL-18 was localized to the intestinal epithelial layer. Anti-IL-18 treatment resulted in a dose-dependent reduction of the severity of colitis in both BALB/c and C57BL/6 mice. Colon shortening following DSS-induced colitis was partially prevented in the treatment groups. In the colon tissue homogenates, IFN-gamma concentrations were lower in the anti-IL-18-treated DSS-fed mice compared with untreated DSS-fed mice. This suppressive effect of anti-IL-18 administered in vivo was also observed on spontaneous tumor necrosis factor-alpha, IL-18, and IFN-gamma production from ex vivo colon organ cultures. The stimulation of lamina propria mononuclear cells by IL-18 and IL-12 resulted in a synergistic increase in IFN-gamma synthesis. These findings suggest that IL-18 is a pivotal mediator in experimental colitis.     

B7 interactions with CD28 and CTLA-4 control tolerance or induction of mucosal inflammation in chronic experimental colitis

CD28-B7 interaction plays a critical costimulatory role in inducing T cell activation, while CTLA-4-B7 interaction provides a negative signal that is essential in immune homeostasis. Transfer of CD45RB(high)CD4(+) T cells from syngeneic mice induces transmural colon inflammation in SCID recipients. This adoptive transfer model was used to investigate the contribution of B7-CD28/CTLA-4 interactions to the control of intestinal inflammation. CD45RB(high)CD4(+) cells from CD28(-/-) mice failed to induce mucosal inflammation in SCID recipients. Administration of anti-B7.1 (but not anti-B7.2) after transfer of wild-type CD45RB(high)CD4(+) cells also prevented wasting disease with colitis, abrogated leukocyte infiltration, and reduced production of proinflammatory cytokines IL-2 and IFN-gamma by lamina propria CD4(+) cells. In contrast, anti-CTLA-4 treatment led to deterioration of disease, to more severe inflammation, and to enhanced production of proinflammatory cytokines. Of note, CD25(+)CD4(+) cells from CD28(-/-) mice similar to those from the wild-type mice were efficient to prevent intestinal mucosal inflammation induced by the wild-type CD45RB(high) cells. The inhibitory functions of these regulatory T cells were effectively blocked by anti-CTLA-4. These data show that the B7-CD28 costimulatory pathway is required for induction of effector T cells and for intestinal mucosal inflammation, while the regulatory T cells function in a CD28-independent way. CTLA-4 signaling plays a key role in maintaining mucosal lymphocyte tolerance, most likely by activating the regulatory T cells.     

Human CD4+ T cells present within the microenvironment of human lung tumors are mobilized by the local and sustained release of IL-12 to kill tumors in situ by indirect effects of IFN-gamma

By implanting nondisrupted pieces of human lung tumor biopsy tissues into SCID mice, it has been possible to establish viable grafts of the tumor, as well as the tumor-associated microenvironment, including inflammatory cells, fibroblasts, tumor vasculature, and the extracellular matrix. Using this xenograft model, we have evaluated and characterized the effects of a local and sustained release of human rIL-12 (rhIL-12) from biodegradable microspheres. In response to rhIL-12, the human CD45+ inflammatory cells present within the xenograft mediate the suppression or the complete arrest of tumor growth in SCID mice. Analysis of the cellular events reveals that human CD4+ and CD8+ T cells are induced by rhIL-12 to produce and secrete IFN-gamma. Serum levels of human IFN-gamma in mice bearing rhIL-12-treated tumor xenografts correlate directly with the degree of tumor suppression, while neutralizing Abs to human IFN-gamma abrogate the IL-12-mediated tumor suppression. Gene expression profiling of tumors responding to intratumoral rhIL-12 demonstrates an up-regulation of IFN-gamma and IFN-gamma-dependent genes not observed in control-treated tumors. Genes encoding a number of proinflammatory cytokines, chemokines (and their receptors), adhesion molecules, activation markers, and the inducible NO synthase are up-regulated following the introduction of rhIL-12, while genes associated with tumor growth, angiogenesis, and metastasis are decreased in expression. NO contributes to the tumor killing because an inhibitor of inducible NO synthase prevents IL-12-induced tumor suppression. Cell depletion studies reveal that the IL-12-induced tumor suppression, IFN-gamma production, and the associated changes in gene expression are all dependent upon CD4+ T cells.     

Blockade of NKG2D signaling prevents the development of murine CD4+ T cell-mediated colitis

It has been recently demonstrated that NKG2D is an activating costimulatory receptor on natural killer (NK) cells, natural killer T (NKT) cells, activated CD8(+) T cells, and gammadelta T cells, which respond to cellular stress, such as inflammation, transformation, and infection. Here we show that intestinal inflammation in colitic SCID mice induced by adoptive transfer of CD4(+)CD45RB(high) T cells is characterized by significant increase of CD4(+)NKG2D(+) T cells and constitutive expression of NKG2D ligands, such as H60, Mult-1, and Rae-1, by lamina propria CD11c(+) dendritic cells. Furthermore, treatment with nondepleting and neutralizing anti-NKG2D MAb after transfer of CD4(+)CD45RB(high) T cells into SCID mice significantly suppressed wasting disease with colitis, abrogated leukocyte infiltration, and reduced production of IFN-gamma by lamina propria CD4(+) T cells. These findings demonstrate that NKG2D signaling pathway is critically involved in CD4(+) T cell-mediated disease progression and suggest a new therapeutic target for inflammatory bowel diseases.     

Expression and release of IL-18 binding protein in response to IFN-gamma.

IL-18 and IL-18 binding protein (IL-18BP) are two newly described opponents in the cytokine network. Local concentrations of these two players may determine biological functions of IL-18 in the context of inflammation, infection, and cancer. As IL-18 appears to be involved in the pathogenesis of Crohn's disease and may modulate tumor growth, we investigated the IL-18/IL-18BPa system in the human colon carcinoma/epithelial cell line DLD-1. In this study, we report that IFN-gamma induces expression and release of IL-18BPa from DLD-1 cells. mRNA induction and secretion of IL-18BPa immunoreactivity were associated with an activity that significantly impaired release of IFN-gamma by IL-12/IL-18-stimulated PBMC. Inducibility of IL-18BPa by IFN-gamma was also observed in LoVo, Caco-2, and HCT116 human colon carcinoma cell lines and in the human keratinocyte cell line HaCaT. Induction of IL-18BPa in colon carcinoma/epithelial cell lines was suppressed by coincubation with sodium butyrate. IFN-gamma-mediated IL-18BPa and its suppression by sodium butyrate were confirmed in organ cultures of intestinal colonic biopsy specimens. In contrast, sodium butyrate did not modulate expression of IL-18. The present data suggest that IFN-gamma may limit biological functions of IL-18 at sites of colonic immune activation by inducing IL-18BPa production. Down-regulation of IL-18BPa by sodium butyrate suggests that reinforcement of local IL-18 activity may contribute to actions of this short-chain fatty acid in the colonic microenvironment.     

Mycophenolate mofetil promotes down-regulation of expanded B cells and production of TNF-alpha in an experimental murine model of colitis

In this study, we used a murine intestinal inflammation model that mimics immunologic characteristics of human Crohn's disease (CD) to investigate the anti-inflammatory effects of mycophenolate mofetil (MMF) on intestinal injury and tissue inflammation. When these colitic mice were pretreated with MMF, we observed a significant decrease in mortality rates and body weight loss as well as an improvement in both wasting and histopathologic signs of colonic inflammation, relative to untreated colitic mice. To determine the mechanisms of action of MMF, we compared various immunological characteristics of the untreated and MMF-pretreated colitic mice. MMF-pretreated colitic mice showed an 18% decrease in the proportion of CD19+ B cells compared with untreated colitic mice 3 days. As a result, MMF pretreatment increases proportion of apoptotic T and B cells, especially CD19+ B cells. Also, down-regulation of Th1 cytokines (TNF-alpha, IFN-gamma) and augmentation of CD4+CD45RB(low) regulatory T (Treg) cells were observed in MMF-pretreated colitic mice compared with untreated colitic mice. Furthermore, mycophenolic acid (MPA) reduced TNF-alpha-stimulated NF-kappaB activation in HT-29 colon epithelial cells. Also, MMF-pretreated colitic mice significantly reduced expression of MD-1 compared with untreated colitic mice on B cells and dendritic cells (DCs). These studies show that MMF pretreatment can improve experimental colitis by down-regulation of expanded B cells population through apoptosis and augmentation of Treg cells. Through these mechanisms, MMF might also be an effective agent for the treatment of other diseases characterized by mucosal inflammation.     

Ameliorating effect of anti-Fas ligand MAb on wasting disease in murine model of chronic colitis

Fas/Fas ligand (FasL) interaction has been implicated in the pathogenesis of various diseases. To clarify the involvement of Fas/FasL in the pathogenesis of intestinal inflammation, we investigated the preventive and therapeutic effects of neutralizing anti-FasL monoclonal antibody (MAb) on the development of chronic colitis induced by adaptive transfer of CD4+CD45RBhigh T cells to SCID mice. Administration of anti-FasL MAb from 1 day after T cell transfer (prevention study) resulted in a significant improvement of clinical manifestations such as wasting and diarrhea. However, histological examination showed that mucosal inflammation in the colon, such as infiltration of T cells and macrophages, was not improved by the anti-FasL MAb treatment. In vitro studies showed that anti-FasL MAb did not inhibit IFN-gamma production by anti-CD3/CD28-stimulated lamina propria CD4+ T cells but suppressed TNF-alpha and IL-1beta production by lamina propria mononuclear cells. Therapeutic administration of anti-FasL MAb from 3 wk after T cell transfer also improved ongoing wasting disease but not intestinal inflammation. These results suggest that the Fas/FasL interaction plays a critical role in regulating systemic wasting disease but not local intestinal inflammation.     

Limited effects of dietary curcumin on Th-1 driven colitis in IL-10 deficient mice suggest an IL-10-dependent mechanism of protection

Curcumin (diferulolylmethane) demonstrates profound anti-inflammatory effects in intestinal epithelial cells (IEC) and in immune cells in vitro and exhibits a protective role in rodent models of chemically induced colitis, with its presumed primary mechanism of action via inhibition of NF-kappaB. Although it has been demonstrated effective in reducing relapse rate in ulcerative colitis patients, curcumin's effectiveness in Crohn's disease (CD) or in Th-1/Th-17 mediated immune models of CD has not been evaluated. Therefore, we investigated the effects of dietary curcumin (0.1-1%) on the development of colitis, immune activation, and in vivo NF-kappaB activity in germ-free IL-10(-/-) or IL-10(-/-);NF-kappaB(EGFP) mice colonized with specific pathogen-free microflora. Proximal and distal colon morphology showed a mild protective effect of curcumin only at 0.1%. Colonic IFN-gamma and IL-12/23p40 mRNA expression followed similar pattern ( approximately 50% inhibition at 0.1%). Secretion of IL-12/23p40 and IFN-gamma by colonic explants and mesenteric lymph node cells was elevated in IL-10(-/-) mice and was not decreased by dietary curcumin. Surprisingly, activation of NF-kappaB in IL-10(-/-) mice (phospho-NF-kappaBp65) or in IL-10(-/-);NF-kappaB(EGFP) mice (whole organ or confocal imaging) was not noticeably inhibited by curcumin. Furthermore, we demonstrate that IL-10 and curcumin act synergistically to downregulate NF-kappaB activity in IEC and IL-12/23p40 production by splenocytes and dendritic cells. In conclusion, curcumin demonstrates limited effectiveness on Th-1 mediated colitis in IL-10(-/-) mice, with moderately improved colonic morphology, but with no significant effect on pathogenic T cell responses and in situ NF-kappaB activity. In vitro studies suggest that the protective effects of curcumin are IL-10 dependent.     

Interleukin-10-independent anti-inflammatory actions of glucagon-like peptide 2

Glucagon-like peptide 2 (GLP-2) is an important intestinal growth factor with anti-inflammatory activity. We hypothesized that GLP-2 decreases mucosal inflammation and the associated increased epithelial proliferation by downregulation of Th1 cytokines attributable to reprogramming of lamina propria immune regulatory cells via an interleukin-10 (IL-10)-independent pathway. The effects of GLP-2 treatment were studied using the IL-10-deficient (IL-10(-/-)) mouse model of colitis. Wild-type and IL-10(-/-) mice received saline or GLP-2 (50 microg/kg sc) treatment for 5 days. GLP-2 treatment resulted in significant amelioration of animal weight loss and reduced intestinal inflammation as assessed by histopathology and myeloperoxidase levels compared with saline-treated animals. In colitis animals, GLP-2 treatment also reduced crypt cell proliferation and crypt cell apoptosis. Proinflammatory (IL-1beta, TNF-alpha, IFN-gamma,) cytokine protein levels were significantly reduced after GLP-2 treatment, whereas IL-4 was significantly increased and IL-6 production was unchanged. Fluorescence-activated cell sorting analysis of lamina propria cells demonstrated a decrease in the CD4(+) T cell population following GLP-2 treatment in colitic mice and an increase in CD11b(+)/F4/80(+) macrophages but no change in CD25(+)FoxP3 T cells or CD11c(+) dendritic cells. In colitis animals, intracellular cytokine analysis demonstrated that GLP-2 decreased lamina propria macrophage TNF-alpha production but increased IGF-1 production, whereas transforming growth factor-beta was unchanged. GLP-2-mediated reduction of crypt cell proliferation was associated with an increase in intestinal epithelial cell suppressor of cytokine signaling (SOCS)-3 expression and reduced STAT-3 signaling. This study shows that the anti-inflammatory effects of GLP-2 are IL-10 independent and that GLP-2 alters the mucosal response of inflamed intestinal epithelial cells and macrophages. In addition, the suggested mechanism of the reduction in inflammation-induced proliferation is attributable to GLP-2 activation of the SOCS-3 pathway, which antagonizes the IL-6-mediated increase in STAT-3 signaling.     

Colitis-inducing potency of CD4+ T cells in immunodeficient, adoptive hosts depends on their state of activation, IL-12 responsiveness, and CD45RB surface phenotype

We studied the induction, severity and rate of progression of inflammatory bowel disease (IBD) induced in SCID mice by the adoptive transfer of low numbers of the following purified BALB/c CD4+ T cell subsets: 1) unfractionated, peripheral, small (resting), or large (activated) CD4+ T cells; 2) fractionated, peripheral, small, or large, CD45RBhigh or CD45RBlow CD4+ T cells; and 3) peripheral IL-12-unresponsive CD4+ T cells from STAT-4-deficient mice. The adoptive transfer into SCID host of comparable numbers of CD4+ T cells was used to assess the colitis-inducing potency of these subsets. Small CD45RBhigh CD4+ T lymphocytes and activated CD4+ T blasts induced early (6-12 wk posttransfer) and severe disease, while small resting and unfractionated CD4+ T cells or CD45RBlow T lymphocytes induced a late-onset disease 12-16 wk posttransfer. SCID mice transplanted with STAT-4-/- CD4+ T cells showed a late-onset IBD manifest > 20 wk posttransfer. In SCID mice with IBD transplanted with IL-12-responsive CD4+ T cells, the colonic lamina propria CD4+ T cells showed a mucosa-seeking memory/effector CD45RBlow Th1 phenotype abundantly producing IFN-gamma and TNF-alpha. In SCID mice transplanted with IL-12-unresponsive STAT-4-/- CD4+ T cells, the colonic lamina propria, mesenteric lymph node, and splenic CD4+ T cells produced very little IFN-gamma but abundant levels of TNF-alpha. The histopathologic appearance of colitis in all transplanted SCID mice was similar. These data indicate that CD45RBhigh and CD45RBlow, IL-12-responsive and IL-12-unresponsive CD4+ T lymphocytes and lymphoblasts have IBD-inducing potential though of varying potency.