Published papers

Inflammatory bowel diseases are thought to be caused by a complex interaction of genetic, immunological, and environmental factors. The involvement of immunological factors in the etiology of inflammatory bowel diseases is suggested by various facts, such as 1) the effectiveness of immunosuppressive agents, steroids, and anti-TNF-α antibody; 2) the presence of autoantibodies, and 3) the spontaneous development of chronic colitis in mice lacking a single immunity-related gene, e.g., IL-2 or TGF-β. In particular, the treatment of Crohn's disease using anti-TNF-α antibody has been applied to clinical practice with satisfactory clinical results all over the world. This article describes the immunological pathology of inflammatory bowel diseases as autoimmune diseases, and outlines prospective new treatment methods based on the nature of the disease.

Background. Apoptosis is a crucial element in the behavior of mammalian cells in many different situations. We here report the establishment of a novel monoclonal antibody (anti-LHK mAb) that has apoptosis-inducing activity against colon cancer Colo205 cells. Methods. The mechanism of anti-LHK mAb-induced cell death was assessed by microscopic morphology, Annexin V/Hoechst 33528 staining, and detection of DNA fragmentation. The molecular weight of LHK antigen was determined by Western blotting. Growth inhibition of Colo205 cells induced by anti-LHK mAb was determined by in vitro and in vivo studies. Results. Anti-LHK reacted with a 70-kDa antigen and completely blocked the proliferation of Colo205 cells bearing LHK in vitro in a manner characteristic of apoptosis. Strikingly, anti-LHK mAb suppressed tumor growth in a murine peritoneal dissemination model. Conclusions. LHK antigen, which is restricted to epithelial cells, may be a novel death receptor that plays a critical role in controlling the growth, invasion, and metastasis of human colon cancer cells. © Springer-Verlag Tokyo 2005.

Naturally arising CD4+CD25+ regulatory T (TR) cells are engaged in the maintenance of self tolerance and prevention of autoimmune diseases. However, accumulating evidence suggests that a fraction of peripheral CD4+CD25- T cells also possesses regulatory activity. Programmed death-1 (PD-1) is a new member of the CD28/CTLA-4 family, which has been implicated in the maintenance of peripheral self tolerance. Here, we identified a subpopulation of CD4+CD25-PD-1+ T cells in the spleen of naive mice that constitutively expressed CTLA-4 and FoxP3 and was hypoproliferative in response to anti-CD3 antibody stimulation in vitro. However, the CD4+CD25-PD-1+ T cells uniquely produced large amounts of IL-4 and IL-10 in response to anti-CD3 and anti-CD28 mAb stimulation, unlike the CD4+CD25+ TR cells. The CD4+CD25-PD-1+ T cells exhibited a suppressor activity against the proliferation of anti-CD3 antibody-stimulated CD4+CD25-PD-1- T cells in vitro, which was partially abrogated by anti-CTLA-4 mAb, but not by anti-IL-10 or anti-PD-1 mAb. Remarkably, the CD4+CD25-PD-1+ T cells inhibited the development of colitis induced by adoptive transfer of CD4+CD45RBhigh T cells into C.B17-scid/scid mice, albeit to a lesser extent than CD4+CD25+ TR cells, in a CTLA-4-dependent manner. These results indicate that the CD4+CD25-PD-1+ T cells contain substantial amounts of TR cells that are involved in the maintenance of peripheral tolerance. © 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Background & Aims: We have previously reported that bone marrow (BM)-derived cells contribute to the regeneration of the human intestinal epithelium. To analyze further how these cells arise, proliferate, and differentiate as epithelial cells, histologic analysis was conducted using endoscopic specimens. Methods: Thirty biopsy specimens from 14 female, sex-mismatched BM-transplantation recipients were examined. BM-derived cells were identified by fluorescent in situ hybridization (FISH) for the Y chromosome and immunohistochemistry. Multicolor FISH was used to exclude cell fusion. These cells were further analyzed for various differentiation or proliferation markers. Results: No evidence of cell fusion was detected. BM-derived cells did not distribute within the crypt as stem cells and rarely expressed Musashi-1. However, BM-derived epithelial cells frequently expressed Ki-67, and some of these cells appeared as pairs of adjacent cells. These cells also expressed markers of all 4 lineages of terminally differentiated cells. During regeneration following graft-vs-host disease, the number of BM-derived cells was substantially increased within Ki-67-positive cells. Interestingly, the number of cells expressing markers for secretory lineage cells was significantly increased within BM-derived cells. This change was unique for BM-derived cells, resulting in a significantly increased proportion of BM-derived cells among secretory lineage cells. Conclusions: BM-derived epithelial cells arise via a mechanism other than cell fusion and rarely give rise to stem cells. However, a small proportion of these cells express proliferation markers, and a majority reside as terminally differentiated cells. During regeneration BM-derived cells increase as secretory lineage cells, thereby contributing to restore epithelial functions. © 2005 by the American Gastroenterological Association.

An immunoproteasome subunit low molecular weight protein 7 (LMP7) plays critical roles in major histocompatibility complex class I antigen processing; however, the mechanism for its expression has remained unclear. We demonstrate that interferon (IFN) regulatory factor-1 (IRF-1) has a pivotal role in IFN-γ-dependent LMP7 expression, as was shown for the other two immunosubunits. A tetracycline-inducible system for IRF-1 revealed its function in the LMP7 expression, and a genomic region functionally interacting with IRF-1 was also determined. Furthermore, the role of IRF-1 in IFN-γ-inducible LMP7 transcription was confirmed by employing small interfering RNA experiments and IRF-1-/- mice. These results suggest that IRF-1 acts as a master regulator for the concerted expression of immunoproteasome components. © 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

We have previously demonstrated that mucosal CD4+ T cells expressing high levels of IL-7 receptor (IL-7Rhigh) are pathogenic cells responsible for chronic colitis. Here we investigate whether IL-7 is directly involved in the expansion of IL-7Rhigh memory CD4 + mucosal T cells and the exacerbation of colitis. We first showed that CD4+ lamina propria lymphocytes (LPLs) from wild-type, T cell receptor-α-deficient (TCR-α-/-), and recombinase- activating gene (RAG)-2-/--transferred mice with or without colitis showed phenotypes of memory cells, but only CD4+ LPLs from colitic mice showed IL-7Rhigh. In vitro stimulation by IL-7, but not by IL-15 and thymic stromal lymphopoietin, enhanced significant proliferative responses and survival of colitic CD4+, but not normal CD4+ LPLs. Importantly, in vivo administration of IL-7 mice accelerated the expansion of IL-7Rhigh memory CD4+ LPLs and thereby exacerbated chronic colitis in RAG-2-/- mice transferred with CD4+ LPLs from colitic TCR-α-/- mice. Conversely, the administration of anti-IL-7R monoclonal antibody significantly inhibited the development of TCR-α-/- colitis with decreased expansion of CD4+ LPLs. Collectively, the present data indicate that IL-7 is essential for the expansion of pathogenic memory CD4+ T cells under pathological conditions. Therefore, therapeutic approaches targeting the IL-7R pathway may be feasible in the treatment of human inflammatory bowel disease. Copyright © 2005 the American Physiological Society.

As our understanding of the immunological and genetic basis of inflammatory bowel disease (IBD) grows, potential therapeutic options are being developed at a rapid pace. Nevertheless, new drugs for IBD are needed because about half of all patients with severe ulcerative colitis (UC) eventually undergo colectomy, and a significant part of Crohn's disease (CD) patients do not respond to standard medical therapies, including immunosuppressants and TNF-α neutralising antibodies, or suffer from significant side effects. Finally, recurrence of disease activity following remission is frequent in both UC and CD, and there is an unmet need for effective maintenance strategies. It is well-known that immune responses in the intestine remain in a state of controlled inflammation, suggesting that not only does active suppression by regulatory T (TR) cells play an important role in the normal intestinal homeostasis, but also its dysregulation might lead to the development of IBD. TR cells are functional subsets of T cells that downregulate adaptive immune responses by interfering with the activation of dendritic cells and proliferation of T cells. From experimental work it is now clear that TR cells play a critical role in maintaining immune homeostasis, and several therapeutic approaches have been targeted at the induction of T R cells in order to control mucosal inflammation. Before using T R cells clinically as living immunosuppressants for the treatment of IBD, however, we have to pass many critical checkpoints, such as the in vitro expansion of TR cells and the confirmation of their safety. This paper will discuss recently gained knowledge of human TR cells and the possibility of their clinical usages as a new strategy for the treatment of IBD. © 2005 Ashley Publications Ltd.

Background: Gut commensal microbes affect the development and activation of the mucosal and systemic immune systems. However, the exact molecular mechanism of these microbes that is involved in the development of colitis remains unclear. Methods: The present study was conducted to determine the distinct role of the innate immune system in the development of a dextran sulfate sodium (DSS) colitis model in MyD88-/- mice, because myeloid differentiation protein (MyD88) is a major adaptor molecule essential for signaling via Toll-like receptors (TLRs). To this end, MyD88-/- and wild-type (WT) mice received sterile distilled water containing 1.2% DSS for 8 days. The survival rate, total clinical score (body weight loss, stool consistency, and rectal bleeding), colon length, and histological score were assessed. The expression of surface markers (F4/80 and CD4) on infiltrating lamina propria mononuclear cells was analyzed immunohistochemistrically. Results: MyD88-/- mice exhibited increased susceptibility to DSS-induced colitis, as reflected by significantly higher lethality and higher clinical and histological scores, and more severe colonic shortening compared to WT mice. Immunohistochemical analysis revealed a significant increase of both F4/80+ macrophages and CD4+ T cells in the inflamed mucosa in DSS-fed MyD88-/- mice compared to DSS-fed WT mice. Conclusions: These findings suggest that, via MyD88 signaling, the innate immune system in the gut plays an important protective role in colitis. © Springer-Verlag Tokyo 2005.

Crohn's disease and ulcerative colitis are two major forms of human inflammatory bowel diseases (IBD). Their etiology is unknown, but increasing evidence indicate that immune mechanisms play an important role. 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 (TR) cells plays an important role in the normal intestinal homeostasis, but also its dysregulation leads to the development of IBD. This article reviews the unique aspects of TR cells and discuss how these control the intestinal homeostasis.

Background and Aims: The authors have previously shown that a third member of the CD28 family, inducible costimulator (ICOS), was increased in the inflamed intestinal mucosa of murine experimental colitis, and that the blockade of ICOS ameliorated the development of colitis. However, the role of ICOS in rat intestinal inflammation and its expression profile remains unclear. In the present study, the authors investigated the involvement of ICOS in the development of rat dextran sulfate sodium (DSS)-induced colitis, and the therapeutic potential of anti-ICOS monoclonal antibody (mAb) in colitis. Methods: The authors first examined expression of ICOS protein in normal rat by immunohistochemistry and flow cytometry. Sprague-Dawley rats were fed 3.0% DSS. The expression of ICOS on infiltrating lamina propria mononuclear cells and splenocytes were examined. The DSS-fed rats were then administered anti-ICOS mAb to test its effect on the development of colitis. Results: Unlike mice and human, ICOS was expressed on a part of CD4+ T-cells from the thymus, spleen, mesenteric lymph nodes and lamina propria. Levels of ICOS on CD4 + T-cells from the spleen and colonic lamina propria were significantly upregulated after Concanavalin A (Con A) stimulation. In addition, ICOS was also upregulated on CD4+ T-cells from DSS-fed rats compared with those from non DSS-fed rats. However, anti-ICOS mAb did not ameliorate the development of both acute and chronic DSS colitis. Conclusion: These results suggest that the different expression of ICOS in rats plays a distinct role in rat intestinal inflammation. © 2004 Blackwell Publishing Asia Pty Ltd.

Background and aims: Many lines of evidence suggest that T helper cell type 1 (Th1) immune responses predominate in Crohn's disease (CD). Recently, a novel transcription factor T-box expressed in T cells (T-bet) has been reported as the master regulator of Th1 development. This study was designed to investigate the role of T-bet and proinflammatory cytokines in Th1 mediated immunopathology in CD. Materials: CD4+ lamina propria mononuclear cells (LPMCs) were isolated from surgically resected specimens (CD, n = 10; ulcerative colitis (UC), n = 10; normal controls (NL), n = 5). Methods: (1) T-bet expression of CD4+ LPMCs was examined by quantitative real time polymerase chain reaction and western blotting. (2) T-bet expression of LPMCs stimulated by interleukin (IL)-12/IL-18 was analysed by western blotting. (3) Interferon γ (IFN-γ) production and T-bet expression of CD4+ peripheral blood mononuclear cells (PBMCs) were examined with or without stimulation by anti-CD3/CD28 monoclonal antibodies and/or IL-12. Results: (1) T-bet expression of CD4+ LPMCs was increased in CD compared with UC and NL. (2) Synergistically, augmentation of IFN-γ production by IL-12/IL-18 was independent of T-bet expression in LPMCs. (3) T-bet was induced by T cell receptor stimulation in CD4+ PBMCs. T-bet induction correlated with IFN-γ production and with augmentation of surface expressed IL-12 receptor β2. Conclusions: T-bet induction by antigenic stimulation and subsequent stimulation by macrophage derived IL-12/IL-18 are important for establishing Th1 mediated immunopathology in CD.

To understand the role of thymic epithelial cells in the development of immature thymocytes, the establishment and cloning of thymic epithelial cell lines must be required. In the present study, we established human and mouse thymic epithelial cell lines through the immortalization by the transfection of cDNA sequences of adenoviral E1a 12S mRNA. This procedure resulted in the isolation of five stable cell lines (one human cell line and four mouse cell lines). These cell lines were positive in cytokeratin demonstrated by immunohistochemistry. Electron microscopic study revealed that they had bundles of tonofilaments and desmosome-like tight junctions. These findings indicate that the cell lines immortalized by E1a gene have retained the properties of epithelial cells. MHC class II antigens were not expressed on these cell lines. When interferon-γ was added to the cultured epithelial cell lines, MHC class II antigens were induced in their cytoplasm and on their surface membrane, demonstrated by immunohistochemical and immunofluorescent studies. It is suggested that these stable cell lines from human and mouse thymus might serve a good tool for the further study of thymocytes differentiation and of unknown cytokines derived from thymus epithelium.

We investigated the in vitro immunosuppressive effect of the sera and the culture supernatants of the peripheral blood mononuclear cells obtained from patients with Crohn's disease. Sera from Crohn's disease markedly suppressed the proliferative response of mouse spleen cells, compared with sera from ulcerative colitis, intestinal tuberculosis and normal controls. The culture supernatants of the adherent mononuclear cells from Crohn's disease showed a remarkable suppressive effect (96±1%), while the culture supernatants of the nonadherent mononuclear cells ha no suppressive activity (-10±16%). The culture supernatants of the adherent and non-adherent mononuclear cells from normal controls had no suppressive activity. The fractionization of the culture supernatants of the adherent cells from the Crohn's disease patients demonstrated that the fractions with high suppressive activity had similar or identical biochemical properties to the serum immunosuppressive fractions which has been reported previously. These results indicate that monocytes may release the serum immunosuppressive factor in Crohn's disease. This factor may contribute to the depression of the immune reactivity in the mucosal lesion and to the persistence of the stimulation of abnormal immune response in the intestinal wall.

Background and Aims: Costimulatory signals are essential for T-cell activation. The aim of this study was to demonstrate expression of costimulatory molecules CD86 and CD80 in human colonic epithelial cells and assess their functional roles in the activation of T cells in inflamed colonic mucosa. Methods: CD86 and CD80 expression was assessed by immunohistochemistry of colonic mucosa, reverse-transcription polymerase chain reaction, and flow cytometric analysis of isolated colonic epithelial cells and cell lines. Costimulatory effect of CD86-expressing colonic epithelial cells on purified CD4+ T-cell proliferation was also assessed at suboptimal phytohemagglutinin stimulation. Results: CD86 and CD80 messenger RNA was detected in isolated colonic epithelial cells from normal and inflamed mucosa of patients with ulcerative colitis. Immunohistochemistry and flow cytometric analysis of colonic epithelial cells confirmed cell surface expression of CD86 protein in inflamed colonic mucosa. Cell surface expression of CD86 protein was increased in the colonic epithelial cell line HT29-18-N2 after interferon gamma stimulation. Purified CD4+ T cells proliferated in response to suboptimal phytohemagglutinin costimulated with interferon gamma-stimulated HT2918-N2, and these proliferative responses were efficiently inhibited by the addition of anti-CD86 monoclonal antibody. Costimulatory effect of CD86-expressing colonic epithelial cells isolated from inflamed mucosa of ulcerative colitis was also demonstrated at suboptimal phytohemagglutinin stimulation in CD4+ T cells. Conclusions: Colonic epithelial cells may act as antigen-presenting cells, and contribute to the activation of T cells through costimulatory molecule CD86 expression in inflamed colonic mucosa.

Persistent severe inflammation in colonic mucosa is thought to cause the development of colon cancer in patients with ulcerative colitis (UC). However, predisposing genetic abnormalities have not been identified in this sequence. Using differential display PCR, we isolated cDNA fragments corresponding to mRNAs that were differentially expressed in colitis- associated cancer tissues and mucosa with mild inflammation in the colon of five UC patients. This molecular screening approach identified 60 cDNA fragments, and we sequenced 34 fragments. One cDNA fragment, which is identical to IFN-inducible gene family 1-8U, was strongly expressed in all five UC-associated cancers. 1-8U was also expressed in sporadic colon cancer tissues and colon cancer cell lines, but not in normal mucosa. This gene was strongly expressed in severely inflamed colonic mucosa of UC without colitis- associated colon cancer, although 1-8U expression was not related to the extent and duration of the disease. However, 1-8U was expressed in the colonic mucosa of all patients with chronic, continuously severe inflammation. These results indicated that IFN-inducible gene family 1-8U expression in inflamed colonic mucosa might be used as a preferential marker of colitis-associated colon cancer in UC.

Background and Aim: Cytomegalovirus (CMV) infection is a serious complication following solid organ and bone marrow transplantation (BMT). CMV affects multiple organs and results in considerable morbidity and prolonged hospitalization. But the clinical feature of upper GI lesions in CMV infection after BMT remains unclear. Detection of CMV antigen in peripheral blood leukocytes with immunohistochemical technique (antigenemia) is the most reliable screening method for CMV infection. The antigenemia is, however, not sensitive enough in some cases. The aim of this study is to investigate the clinical significance of upper GI lesions in CMV infection. Patients and Methods: We performed endoscopic examination of the upper GI tract in 47 patients 20 to 70 days after allogeneic BMT from December 1992 to November 1997. Biopsy specimens, taken each from esophagus, stomach body and antrum, and duodenum, were assayed for CMV antigen with immunohistochemical technique. Results: CMV infection was revealed in the upper GI tract in 9 patients. The endoscopic findings of the affected lesions were characteristic erosions surrounded by redness in the stomach and duodenum, map-like redness in the duodenum, and ulcer in the esophagus. Stomach was involved in all cases (9/9, 100%), but duodenum (4/9, 44%) and esophagus (2/9, 22%) were less frequently infected. In 3 patients, CMV antigen was detected in the upper GI tract before antigenemia turned positive. Conclusion: The endoscopic examination of upper GI tract is a useful screening method for CMV infection following allogeneic BMT.