Published papers

It is well known that immune responses in the intestine remain in a state of controlled inflammation, suggesting that not only active suppression by regulatory T cells plays an important role in the normal intestinal homeostasis, but also its dysregulation leads to the development of inflammatory bowel disease. In this study, we demonstrate that the CD4+CD25 bright T cells reside in the human intestinal lamina propria (LP) and functionally retain regulatory activities. All human LP CD4+ T cells regardless of CD25 expression constitutively expressed CTLA-4, glucocorticoid-induced TNFR family-related protein, and Foxp3 and proliferate poorly. Although LP CD4+CD25- T cells showed an activated and anergic/memory phenotype, they did not retain regulatory activity. In LP CD4+CD25+ T cells, however, cells expressing CD25 at high levels (CD4+CD25bright) suppressed the proliferation and various cytokine productions of CD4+CD25- T cells. LP CD4+CD25bright T cells by themselves produced fewer amounts of IL-2, IFN-γ, and IL-10. Interestingly, LP CD4 +CD25bright T cells with regulatory T activity were significantly increased in patients with active inflammatory bowel disease. These results suggest that CD4+CD25bright T cells found in the normal and inflamed intestinal mucosa selectively inhibit the host immune response and therefore may contribute to the intestinal immune homeostasis.

Intestinal epithelial cell-derived interleukin (IL)-7 functions as a pleiotropic and nonredundant cytokine in the human intestinal mucosa; however, the molecular basis of its production has remained totally unknown. We here showed that human intestinal epithelial cells both constitutively and when induced by gamma interferon (IFN-γ) produced IL-7, while several other factors we tested had no effect. Transcriptional regulation via an IFN regulatory factor element (IRF-E) on the 5′ flanking region, which lacks canonical core promoter sequences, was pivotal for both modes of IL-7 expression. IRF-1 and IRF-2, the latter of which is generally known as a transcriptional repressor, were shown to interact with IRF-E and transactivate IL-7 gene expression in an IFN-γ-inducible and constitutive manner, respectively. Indeed, tetracycline-inducible expression experiments revealed that both of these IRF proteins up-regulated IL-7 protein production, and their exclusive roles were further confirmed by small interfering RNA-mediated gene silencing systems. Moreover, these IRFs displayed distinct properties concerning the profile of IL-7 transcripts upon activation and expression patterns within human colonic epithelial tissues. These results suggest that the functional interplay between IRF-1 and IRF-2 serves as an elaborate and cooperative mechanism for timely as well as continuous regulation of IL-7 production that is essential for local immune regulation within human intestinal mucosa.

Background/Aims: Recent advances in molecular cloning of hepatitis C virus (HCV) have enabled us to apply some available HCV molecular clones to experimental studies. However, these investigations have been restricted to chimpanzee models or 'isolated hepatocytes' from tree shrews. In this study, we engrafted 'human liver tissue' into immunodeficient mice and investigated HCV infection using an infectious molecular clone. Methods: Human liver tissues from normal (non-HCV-infected) liver were transplanted into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. We then inoculated the mice with sera from HCV-infected patients or an infectious HCV molecular clone. HCV RNA was assessed using nested reverse-transcription polymerase chain reaction (PCR), real-time detection PCR and in situ PCR. Results: Without any growth support, normal human liver tissues survived in NOD/SCID mice while maintaining the original viable hepatic architecture. HCV RNA was detected in the mice serum until the fourth week after the inoculation. In situ PCR and immunohistochemistry clearly demonstrated positive signals for HCV in the cytoplasm of infected hepatocytes, while the engrafted human liver tissues showed no apparent morphological changes indicative of infection. Conclusion: Engraftment of human liver tissues into NOD/SCID mice and infection with HCV molecular clones could offer a reverse genetic strategy for HCV infection. Copyright © Blackwell Munksgaard 2004.

Intestinal epithelial cell (IEC)-derived cytokines, such as stem cell factor (SCF), interleukin (IL)-7 and IL-15 are known to be required for the development of intestinal intraepithelial lymphocytes (IELs). A newly described cytokine, IL-18, has also been shown to be produced by intestinal epithelial cells. To demonstrate the functional effects of IL-18 on human IELs, we assessed IL-18/IL-18 receptor expression in IEC/IEL and proliferation following stimulation of intestinal IELs by IL-18. IL-18 transcripts were detected both in freshly isolated human colonie epithelial cells and in various colonie epithelial cell lines. IL-18 protein was also detected by ELISA and flow cytometric analysis using antihuman IL-18-specific monoclonal antibody (MoAb). Furthermore, IELs constitutively expressed the IL-18 receptor in addition to the IL-2 and IL-7 receptors. More importantly, IL-18 augmented significant proliferative responses of IEL in combination with IL-2, IL-7 and IL-15 both in the presence and in absence of anti-CD3 MoAb. These results suggest that IL-18 might play a crucial role in the proliferation and maintenance of intestinal IELs.

Several studies indicate that CD4+ T cells, macrophages, and dendritic cells initially mediate intestinal inflammation in murine models of human inflammatory bowel disease. However, the initial role of B cells in the development of intestinal inflammation remains unclear. In this study we present evidence that B cells can trigger intestinal inflammation using transgenic (Tg) mice expressing CD40 ligand (CD40L) ectopically on B cells (CD40L/B Tg). We demonstrated that CD40L/B Tg mice spontaneously developed severe transmural intestinal inflammation in both colon and ileum at 8-15 wk of age. In contrast, CD40L/B TgxCD40-/- double-mutant mice did not develop colitis, indicating the direct involvement of CD40-CD40L interaction in the development of intestinal inflammation. The inflammatory infiltrates consisted predominantly of massive aggregated, IgM-positive B cells. These mice were also characterized by the presence of anti-colon autoantibodies and elevated IFN-γ production. Furthermore, although mice transferred with CD4+ T cells alone or with both CD4+ T and B220 + B cells, but not B220+ cells alone, from diseased CD40L/B Tg mice, develop colitis, mice transferred with B220+ B cells from diseased CD40L/B Tg mice and CD4+ T cells from wild-type mice also develop colitis, indicating that the Tg B cells should be a trigger for this colitis model, whereas T cells are involved as effectors. As it has been demonstrated that CD40L is ectopically expressed on B cells in some autoimmune diseases, the present study suggests the possible contribution of B cells in triggering intestinal inflammation in human inflammatory bowel disease.

Background & Aims: To investigate the role of inducible costimulator (ICOS), a new member of the CD28 family involved in regulation of T-cell activation and chronic intestinal inflammation, we assessed its expression and functional role in patients with inflammatory bowel disease (IBD). Methods: Expression of ICOS, CD28, and cytotoxic T-lymphocyte antigen (CTLA) 4 on intestinal lamina propria mononuclear cells (LPMC) from patients with ulcerative colitis (UC), Crohn's disease (CD), and normal controls was determined using flow cytometry and immunohistochemistry. Expressions of the ICOS ligand, B7h, on lamina propria B cells, macrophages, and epithelial cells (EC) in the intestinal mucosa were also determined using flow cytometry. The functional costimulatory effect of ICOS on LPMC was assessed by the proliferative response and cytokine production. Results: CD4+ LPMC expressing ICOS was significantly increased in the inflamed mucosa of IBD patients but not in inflammatory or normal controls. B7h was also significantly up-regulated on B cells, macrophages, and EC in inflamed mucosa of IBD patients. Proliferative responses of anti-CD3/ICOS costimulation were significantly higher compared with those of anti-CD3 monoclonal antibody (mAb) alone. Anti-CD3/ICOS-stimulated-LPMC from UC secreted significantly increased amounts of interleukin (IL)-5 among the 3 groups. In contrast, anti-CD3/ICOS-stimulated-LPMC from CD secreted significantly increased amounts of interferon (IFN)-γ in the presence of IL-12. Conclusions: Highly expressed ICOS in activated CD4+ LPMC of IBD patients contributes to the dysregulated immune responses in IBD. Because ICOS hyperexpression was limited to inflammatory sites in IBD patients, ICOS would be a feasible therapeutic target for the treatment of IBD.

Interleukin-7 (IL-7) is an indispensable cytokine for the development of lymphocyte lineage cells. However, the potential role of IL-7 in peripheral nonlymphoid tissues was unclear before our study. We have demonstrated that intestinal epithelial cells produce IL-7 and that IL-7 serves as a regulatory factor for proliferation of mucosal lymphocytes expressing IL-7 receptor (IL-7R). Recent studies demonstrated that intestinal epithelial cell-derived IL-7 plays a crucial role in the organization of mucosal lymphoid tissues and regulating the normal immune response in intestinal mucosa. In a previous study, we demonstrated that IL-7 transgenic mice developed chronic colitis. Here, we have demonstrated the essential role of mucosal IL-7/IL-7R-dependent signals in the development of chronic intestinal inflammation. Our results indicate that mucosal IL-7/IL-7R-dependent signals are involved in the development of chronic intestinal inflammation in both the mouse model and human disease of the intestinal mucosa. Therefore, current studies indicate that therapeutic approaches by specific targeting of IL-7R-expressing mucosal T cells may be feasible in the treatment of human ulcerative colitis.

MyD88 is a key adaptor molecule for signalling via Toll-like receptors (TLRs) and the response to gut commensal microbes. To investigate the role of TLRs/MyD88 pathway in the development of the gut-associated lymphoid tissue (GALT), we examined the development of Peyer's patches (PPs) and cryptopatch (CP), and also one of effector compartment, intraepithelial lymphocyte (IEL) in MyD88 -/-, TLR2 -/- and TLR4 -/- mice. In MyD88 -/- mice, the organogenesis of PPs was not disturbed. However, PPs in 2-week-old MyD88 -/- mice were significantly smaller than those in MyD88 +/- mice. Also, in 2-week-old TLR4 -/-, but not TLR2 -/- mice, PPs did not develop rapidly. The development of PPs in MyD88 -/- and TLR4 -/- mice was completely recovered in 10 weeks. PP cells from MyD88 -/- mice showed significant decrease in proliferation when stimulated with lipopolysaccharide. The development of CP and IEL was also normal in 10-week-old MyD88 -/- mice. These results suggest that the TLRs/MyD88 pathway might be involved in the development of PPs only at early postnatal stage, and TLRs/MyD88-independent signalling is critically involved in the development of GALT in adult mice.

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 +CD45RBhigh 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 +CD45RBhigh T cells suppressed wasting disease with colitis, abrogated leukocyte infiltration, and reduced the production of IFN-γ, IL-2, and TNF-α, 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.

Lympho-haemopoietic progenitors residing in murine gut cryptopatches (CPs) have been shown to generate intestinal extrathymic intraepithelial lymphocytes (IELs). However, the role of CPs in the development of intestinal inflammation remains unclear. To investigate the role of CPs in the development of intestinal inflammation, we examined SAMP1/Yit mice, which spontaneously develop a chronic intestinal inflammation localized to the terminal ileum and cecum. Here, we showed the sharp correlation between the disease onset and the decreased number of CPs, resulting in decreased number of both thymus-independent IELs including T-cell receptor γδ+ (TCRγδ+) and CD8αα+TCRα β+ cells but not thymus-dependent CD8αβ +TCRαβ+ and CD4+TCRαβ + cells in SAMP1/Yit mice. These data provide the first suggestion that thymus-independent IELs derived from CP might play protective role against the onset and the development of intestinal inflammation.

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+CD45RB high 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-γ production by anti-CD3/CD28-stimulated lamina propria CD4 + T cells but suppressed TNF-α and IL-1β 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.

The IL-7/IL-7R-dependent signaling pathway plays a crucial role in regulating the immune response in intestinal mucosa. Here we demonstrate the pivotal role of this pathway in the development and treatment of chronic colitis. T cells expressing high levels of IL-7R were substantially infiltrated in the chronic inflamed mucosa of TCR α-chain knockout mice and IL-7 transgenic mice. Transfer of mucosai T cells expressing high levels of IL-7R, but not T cells expressing low levels of IL-7R, from these mice into recombinase-activating gene-2-/- mice induced chronic colitis. Selective elimination of T cells expressing high levels of IL-7R by administrating small amounts of toxin-conjugated anti-IL-7R Ab completely ameliorated established, ongoing colitis. These findings provide evidence that therapeutic approaches targeting mucosal T cells expressing high levels of IL-7R are effective in the treatment of chronic intestinal inflammation and may be feasible for use in the therapy of human inflammatory bowel disease.

Background and Aim: The authors have previously shown that production of interleukin (IL)-18 was increased in the inflamed mucosa of patients with Crohn's disease (CD) and blockade of IL-18 ameliorated the murine model of CD. This demonstrated that IL-18 plays a significant role during intestinal inflammation. However, the initial role of IL-18 during intestinal inflammation was unclear; therefore the susceptibility of IL-18 transgenic (Tg) mice to acute dextran sulfate sodium (DSS)-induced colitis was examined. Methods: Interleukin-18 Tg and wild-type (WT) mice were fed 2.0% of DSS for 8 days. The total clinical scores (bodyweight loss, stool consistency, and rectal bleeding), colon length and histological scores were assessed. The expressions of surface markers and IL-18 on infiltrating lamina propria mononuclear cells were analyzed immunohistochemistrically. Mesenteric lymph node (MLN) cells were isolated and the expressions of CD4+ T-cell activation markers (CD69, CD25 and IL18R) were analyzed by flow cytometry. Results: The IL-18 Tg mice exhibited an increased susceptibility to DSS-induced colitis, as shown by significantly increased clinical, histological scores, and more severe colonic shortening compared with WT mice. Immunohistochemical analysis revealed a significant increase of IL-18 production and CD11b+ macrophages but not CD4+T cells in the inflamed mucosa in DSS-fed IL-18 Tg compared with DSS-fed WT mice. Furthermore, MLN cells revealed no evidence of increased CD4+T-cell activation in DSS-fed IL-18 Tg. Conclusions: These findings suggest that IL-18 overproduction in the mucosa plays an important role in the marked infiltration of macrophages and exacerbates colitis in IL-18 Tg mice. © 2003 Blackwell Publishing Asia Pty Ltd.

Background: Interleukin (IL)-12 and IL-18 are major interferon (IFN)-γ-inducing factors that collaborate with each other. The present study was conducted to determine the distinct roles of IL-12 and IL-18 in the development of dextran sulphate sodium (DSS) colitis in mice. Methods: Colitis was induced in IL-12p35-/-, IL-18-/-, IL-18 receptor -/- and control mice with DSS. Clinical and histopathological analysis was conducted using survival rate, weight loss score, diarrhoea score, bloody stool score and histological score. In addition, cytokine production by lamina propria mononuclear cells (LPMCs) was examined using the specific enzyme-linked immunoassay. Results: IL-12p35-/- mice developed only a mild disease associated with no lethality and few histopathological abnormalities. In contrast, IL-18-/- and IL-18R-/- mice developed more severe colitis associated with high lethality and more histopathological abnormalities compared with control mice. LPMCs from DSS-fed IL-18-/- mice produced significantly higher amounts of IFN-γ, while LPMCs from DSS-fed IL-12-/- mice produced lower amounts of IFN-γ and tumour necrosis factor (TNF)-α compared with control mice. Conclusion: These results suggest that IL-18 might function with manners different from IL-12 at some pathological conditions in the development of colitis.

CD4+CD25+ regulatory T cells in normal animals are engaged in the maintenance of immunological self-tolerance and prevention of autoimmune disease. However, accumulating evidence suggests that a fraction of the peripheral CD4+CD25- T cell population also possesses regulatory activity in vivo. Recently, it has been shown glucocorticoid-induced TNFR family-related gene (GITR) is predominantly expressed on CD4+CD25+ regulatory T cells. In this study, we show evidence that CD4+GITR+ T cells, regardless of the CD25 expression, regulate the mucosal immune responses and intestinal inflammation. SCID mice restored with the CD4+GITR- T cell population developed wasting disease and severe chronic colitis. Cotransfer of CD4+GITR+ population prevented the development of CD4+CD45RBhigh T cell-transferred colitis. Administration of anti-GITR mAb-induced chronic colitis in mice restored both CD45RBhigh and CD45RBlow CD4+ T cells. Interestingly, both CD4+CD25+ and CD4+CD25- GITR+ T cells prevented wasting disease and colitis. Furthermore, in vitro studies revealed that CD4+CD25-GITR+ T cells as well as CD4+CD25+GITR+ T cells expressed CTLA-4 intracellularly, showed anergic, suppressed T cell proliferation, and produced IL-10 and TGF-β. These data suggest that GITR can be used as a specific marker for regulatory T cells controlling mucosal inflammation and also as a target for treatment of inflammatory bowel disease.

T-cell co-stimulatory molecule, inducible co-stimulator (ICOS)/B7-related protein-1 (B7RP-1) interactions play an essential role of T-cell-dependent B-cell activation in peripheral lymphoid organs such as spleen and lymph nodes. Here, we investigate the role of ICOS/B7RP-1 interactions in the development of Peyer's patches (PPs). In ICOS-/- mice, the number of PPs was not decreased, although PPs in ICOS-/- mice were significantly reduced in size. Phenotypic analysis showed no obvious differences between ICOS-/- and ICOS+/- mice in the distribution of T-cells, B-cells, macrophages and dendritic cells. However, PNA+ cells characteristic of intestinal germinal centers were totally absent in ICOS-/- mice. Moreover, production of IgA and IgG, but not IgM was significantly reduced in PPs in ICOS-/- mice. These data suggest that ICOS/B7RP-1 interactions may not affect the organogenesis, but involve in the functional development of PPs. © 2003 Elsevier Science B.V. All rights reserved.

Various genetic alterations in ulcerative colitis (UC)-associated colorectal cancers (CRCs) have been reported. However, almost all studies have shown abnormalities of the known genes that have been demonstrated in sporadic CRCs. To identify novel genetic alterations, we selected unintentionally 35 microsatellite markers, and performed allelotype study in CRCs or dysplastic lesions from UC. High frequency of loss of heterozygosity (LOH; 62.5%) was detected on chromosome 6 (D6S468) but not on other chromosomes. With four additional microsatellite markers around the D6S468 locus, we determined the commonly deleted region between two loci, D6S1543 and D6S1580, in UC-associated CRCs and dysplasia. Interestingly, there was no LOH in this region in sporadic CRCs and severely inflamed lesions of longstanding and extensive UC without cancers. These results indicated the presence of novel tumor suppressor genes on chromosome 6 related to the carcinogenesis of UC but not to sporadic CSCs.

Crohn's disease is an inflammatory bowel disease associated with several changes in the immune system, including an increased number of infiltrating macrophages. These macrophages release a variety of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha, macrophage infiltrating factor (MIF), interleukin (IL)-6, IL-12 and IL-18, which are critically involved in the onset and the development of Crohn's disease. We here focus on the role of macrophages, especially macrophage-derived IL-18 in both patients with Crohn's disease and a murine model of Crohn's disease.

Inflammatory bowel disease is thought to result from inappropriate activation of mucosal immune responses. Intestinal epithelial cells produce interleukin (IL)-7 that serves as a regulatory factor for IL-7 receptor (IL-7R)(+) mucosal lymphocytes. The pivotal role of mucosal IL-7/IL-7R dependent signals in the activation of mucosal immune responses that lead to the development of chronic intestinal inflammation are demonstrated. Therapeutic approaches targeting IL-7/IL-7R signal pathway may be feasible in the treatment of inflammatory bowel disease.