Published papers

Background: In recent years leukocytapheresis using a leukocyte removal filter (known as lymphocytapheresis, LCAP) has been applied to the treatment of various autoimmune diseases including ulcerative colitis (UC). In the present study we aimed to clarify how LCAP therapy modifies inflammatory responses by modulating circulating TNF-α-producing monocytes. Methods: Mononuclear cells were obtained from blood before and after the first treatment, and the expression profiles of various immune cells (naive versus, memory, regulatory CD4+CD25bright versus non-regulatory CD4 +CD25- T cells, and CD14+CD16- versus CD14dullCD16+ monocytes) were assessed. To evaluate immunological differences between CD14+CD16- and CD14dullCD16+ monocytes, the expression of TNF-α, IL-6, IL-12, IL-10, IL-18, surface toll-like receptor 2 (TLR2), TLR4, and other activation markers including HLA-DR, CD80 and CD86, as well as cytokine profiles, were analyzed. Results: LCAP treatment selectively removed CD14 dullCD16+ monocytes, which preferentially produce TNF-α and IL-12 and express HLA-DR, CD80, CD86, and TLR2, compared with the major fraction of CD14+CD16- monocytes, which conversely produce a higher amount of IL-10. In addition, the CD4 +CD45RO+CD62L-/CD4+CD45RO +CD62L+ ratio was significantly lower after LCAP therapy. However, the CD4+CD25bright/total CD4+ ratio did not change. Conclusions: The present findings revealed the real target of proinflammatory CD14dullCD16+ monocytes removed during LCAP treatment of UC and that LCAP might be used as an extracorporeal anti-TNF-α therapy, expanding the clinical applications of this procedure to include the treatment of Crohn's disease. Copyright © 2006 Crohn's & Colitis Foundation of America, Inc.

A 44-year-old women developed marked myopathy one year earlier, when she was treated with intravenous prednisolone for acute severe exacerbation of ulcerative colitis. When she was admitted to our hospital for another severe exacerbation, intravenous cyclosporine A was administered as monotherapy because she could not tolerate corticosteroid. The treatment was successful and she obtained complete remission. Cyclosporine A monotherapy is considered to be a valuable alternative to proctocolectomy for severe ulcerative colitis patients who cannot tolerate corticosteroid.

Background & Aims: Although bone marrow (BM) is known as a primary lymphoid organ, it also is known to harbor memory T cells, suggesting that this compartment is a preferential site for migration and/or selective retention of memory T cells. We here report the existence and the potential ability to induce colitis of the colitogenic BM CD4+ memory T cells in murine colitis models. Methods: We isolated BM CD4+ T cells obtained from colitic severe combined immunodeficient mice induced by the adoptive transfer of CD4+CD45RBhigh T cells and colitic interleukin (IL)-10-/- mice that develop colitis spontaneously, and analyzed the surface phenotype, cytokine production, and potential activity to induce colitis. Furthermore, we assessed the role of IL-7 to maintain the colitogenic BM CD4+ T cells. Results: A high number of CD4+ T cells reside in the BM of colitic severe combined immunodeficient mice and diseased IL-10-/- mice, and they retain significant potential to induce type-1 T helper-mediated colitis in an IL-7-dependent manner. These resident BM CD4+ T cells have an effector memory (TEM; CD44highCD62L-IL-7Rhigh) phenotype and preferentially are attached to IL-7-producing BM cells. Furthermore, the accumulation of BM CD4+ TEM cells was decreased significantly in IL-7-deficient recipients reconstituted with the colitogenic lamina propria CD4+ TEM cells. Conclusions: Collectively, these findings suggest that BM-retaining colitogenic CD4+ memory T cells in colitic mice play a critical role as a reservoir for persisting lifelong colitis. © 2007 AGA Institute.

Background & Aims: The transcription factor Hath1 plays a crucial role in the differentiation program of the human gut epithelium. The present study was conducted to investigate the molecular mechanism of Hath1 expression and its close association with β-catenin/glycogen synthase kinase 3β (GSK3β) under the Wnt pathway in human colonocytes. Methods: Tissue distribution of Hath1 messenger RNA in human tissues was examined by Northern blot. Stability of Hath1 protein was analyzed by expression of FLAG-tagged Hath1 in human cell lines. Targeting of Hath1 protein by GSK3β was determined by specific inhibition of GSK-3β function. Expression of Hath1 protein in colorectal cancers was examined by immunohistochemistry. Results: Hath1 messenger RNA expression was confined to the lower gastrointestinal tract in human adult tissues. In colon cancer cells, although Hath1 messenger RNA was also detected, Hath1 protein was positively degradated by proteasome-mediated proteolysis. Surprisingly, the GSK3β-dependent protein degradation was switched between Hath1 and β-catenin by Wnt signaling, leading to the dramatic alteration of cell status between proliferation and differentiation, respectively. Hath1 protein was detected exclusively in normal colon tissues but not in cancer tissues, where nuclear-localized β-catenin was present. Conclusions: The present study suggests a novel function of the canonical Wnt signaling in human colon cancer cells, regulating cell proliferation and differentiation by GSK3β-mediated, reciprocal degradation of β-catenin or Hath1, respectively, which further emphasizes the importance of aberrant Wnt signaling in colonocyte transformation. © 2007 AGA Institute.

FTY720, a sphingosine-derived immunomodulator, causes immunosuppression via enhancement of lymphocyte sequestration into secondary lymphoid organs, thereby preventing their antigen-activated T cell egress to sites of inflammation. FTY720 is highly effective in inhibiting autoimmunity in various animal models. However, there is little known about how FTY720 controls the migration property of memory T cells. Here, we demonstrated that FTY720 prevents the development of colitis induced by the adoptive transfer of lamina propria (LP) colitogenic effector memory CD4+ T cells (TEM cells; CD45RB lowCD44highCD62L-) into severe combined immunodeficiency (SCID) mice and suppresses interferon-γ, interleukin-2, and tumor necrosis factor-α production by LP CD4+ T cells. The numbers of spleen, peripheral blood, mesenteric lymph node, and LP CD4 + T cells in FTY720-treated mice were significantly reduced compared with those in control mice. Notably, LP CD4 TEM cells as well as splenic CD4+CD45RBhigh T cells expressed several spingosine-1-phosphate receptors that are targets for FTY720. Furthermore, FTY720 also prevented the development of colitis induced by the adoptive transfer of splenic CD4+CD45RBhigh T cells into SCID mice. Collectively, the present data indicate that FTY720 treatment may offer the potential not only to prevent the onset of disease but also to treat memory T cell-mediated autoimmune diseases including inflammatory bowel diseases. Copyright © 2006 the American Physiological Society.

Background: Crohn's disease (CD) is an inflammatory bowel disease that is associated with several changes in the immune system, including an increased number of infiltrating macrophages. These macrophages release a variety of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) which are critically involved in the onset and the development of CD. The present study was performed to explore the initial involvement of macrophages in the development of T-cell-mediated chronic colitis. Methods: The effects were evaluated of saporin-conjugated anti-CD11b monoclonal antibody (mAb) on the development of chronic colitis in severe combined immunodeficiency (SCID) mice induced by adoptive transfer of CD4+CD45RBhigh T cells as an animal model of CD. Results: Significantly increased CD11b-expressing macrophages as well as CD4+ T cells were found in inflamed colon from colitic mice. Administration of saporin-conjugated anti-CD11b mAb markedly ameliorated the clinical and histopathological disease. In vivo treatment with saporin-conjugated anti-CD11b mAb decreased CD4+ T-cell infiltration in the colon and suppressed inferferon-γ (IFN-γ) and TNF-α production by lamina propria CD4+ T cells. Conclusions: Collectively, the present results suggest an initial role of macrophages in the pathogenesis of T-cell-mediated chronic colitis. Furthermore, the macrophage-specific targeting may be a promising strategy for therapeutic intervention in CD. © 2006 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd.

A 23-year-old woman was admitted in November, 2002, complaining pain of the left side and buttock. She had ulcerative colitis when she was 16 and received medical treatment. Based on physical examination and findings of magnetic resonance imaging and bone scintigrapy, as sacroiliitis complicated by ulcerative colitis, was diagnosed Reports on sacroiliitis and ankylosing spondylitis complicated by inflammatory bowel diseases (IBD) are relatively rare in Japan, whereas they are common complications of IBD in Western countries. The efficacy of steroids on pain relief of sacroiliitis and ankylosing spondylitis is unclear.

A 23-year-old man was admitted for treatment of acute exacerbation of ileitis and perianal abscess caused by Crohn's disease. After incision and drainage of the abscess, coupled with antibiotic therapy, 6-mercaptopurine (6-MP) was commenced. His white blood cell (WBC) count on day 12 after initiation of 6-MP was not decreased. However, on day 24 he was re-admitted because of severe myelosuppression (WBC: 300/μl), which was complicated by the recurrence of the perianal abscess. Myelosuppression was prolonged and required the administration of granulocyte colony stimulating factor (G-CSF). G-CSF was continued for 17 days to achieve recovery of his WBC count to a normal level.

Naturally arising CD4+CD25+ regulatory T (T R) cells have been shown to prevent and cure murine T cell-mediated colitis. However, their exact mechanism of controlling colitogenic memory CD4+ T cells in in vivo systems excluding the initial process of naive T cell activation and differentiation has not been examined to date. Using the colitogenic effector memory (TEM) CD4+ cell-mediated colitis model induced by adoptive transfer of colitogenic CD4 +CD44highCD62L- lamina propria (LP) T cells obtained from colitic CD4+CD45RBhigh T cell-transferred mice, we have shown in the present study that CD4+CD25+ TR cells are able not only to suppress the development of colitis, Th1 cytokine production, and the expansion of colitogenic LP CD4+ TEM cells but also to expand these cells by themselves extensively in vivo. An in vitro coculture assay revealed that CD4+CD25+ TR cells proliferated in the presence of IL-2-producing colitogenic LP CD4+ TEM cells at the early time point (48 h after culture), followed by the acquisition of suppressive activity at the late time point (96 h after culture). Collectively, these data suggest the distinct timing of the IL-2-dependent expansion of CD4+CD25+ T R cells and the their suppressive activity on colitogenic LP CD4 + TEM cells. Copyright © 2006 the American Physiological Society.

Ulcerative colitis (UC) and Crohn's disease (CD) are debilitating idiopathic inflammatory bowel diseases (IBDs) with symptoms that impair ability to function and quality of life. The aetiology of IBD is inadequately understood and, therefore, drug therapy has been empirical instead of based on sound understanding of the disease mechanisms. This has been a major factor for poor drug efficacy and treatment-related side effects that often add to disease complications. The development of biologicals, notably infliximab, to block TNF-α reflects some progress, but there is major concern about their side effects and lack of long-term safety and efficacy profiles. However, IBD by its very nature is exacerbated and perpetuated by inflammatory cytokines, including TNF-α, IL-6 and IL-12, for which activated peripheral blood lymphocytes, monocytes/macrophages and granulocytes are major sources. Hence, activated leukocytes should be appropriate targets of therapy. At present, three strategies are available for removing excess and activated leukocytes by leukocytapheresis: centrifugation, Adacolumn® and Cellsorba™. Centrifugation can deplete lymphocytes or total leukocytes, whereas Adacolumn selectively adsorbs granulocytes and monocytes together with a smaller fraction of lymphocytes (FcγR- and complement receptor-bearing leukocytes), and Cellsorba non-selectively removes all three major leukocyte populations. Efficacy has ranged from 'none' to an impressive 93% together with excellent safety profiles and downmodulation of inflammation factors. Furthermore, leukocytapheresis has shown strong drug-sparing effects and reduced the number of patients requiring colectomy or exposure to unsafe immunosuppressants, such as cyclosporin A. Leukocytapheresis removes from the body cells that contribute to IBD and, therefore, unlike drugs, it is not expected to induce dependency or refractoriness. © 2006 Informa UK Ltd.

Background: Transfer of CD4 +CD45RB high T cells from normal donors to SCID/Rag-1, 2-deficient mice, which lack T and B cells, leads to the development of a T H1-mediated inflammatory bowel disease (IBD)-like syndrome characterized by extensive mononuclear cell infiltrates and epithelial cell hyperplasia. Because it is well known that B cells are also involved in a multitude of mechanistic pathways in human IBD, this study attempts to establish a new model of colitis in nude mice. Methods: We transferred CD4 +CD45RB high T cells into athymic nude mice, which lack thymus-dependent T cells but retain normal B cells, to establish and investigate a B cell-involving chronic colitis model. As a control, CD4 +CD25 - T cells were also used. Results: Mice reconstituted with CD4 +CD45RB high but not CD4 +CD25 - T cells developed a wasting disease, with severe infiltrates of B cell aggregates as well as T cells, macrophages, and dendritic cells into the colon and elevated levels of interferon-γ, tumor necrosis factor-α, interleukin (IL)-4, IL-5, and IL-10, by 7 weeks after T cell transfer. Furthermore, the infiltrated lamina propria B cells in colitic nude mice consisted predominantly of massive aggregated immunoglobulin (Ig) M- and scattered IgG-positive cells, but not IgA-positive cells. In contrast, mice reconstituted with CD4 +CD45RB high and CD4 +CD45RB low did not develop wasting disease or colitis. Conclusions: Collectively, the power of the colitis model induced by the adoptive transfer of CD4 +CD45RB high T cells into nude mice is that one can investigate the roles of T H2-type cells and B cells in a regulatory T cell-depleted condition. Copyright © 2006 by Lippincott Williams & Wilkins.

The appropriate therapy for Crohn's disease (CD) cannot be achieved until the extent of disease and its anatomical importance are taken into consideration. Therefore, information from imaging tests is essential to establish a diagnosis. A single gold standard for the diagnosis of CD is not available. The diagnosis should be made by clinical evaluation and a combination of endoscopic, histological, radiological, and/or biochemical investigations. In the case of patients with suspected CD in whom stenosis is not confirmed, if ileocolonoscopy and radiological examination/cross-sectional imaging show no lesion or they are difficult to conduct, small bowel capsule endoscopy is applicable. Radiological examination, especially, double-contrast imaging of the small intestine is one of the best tools for the evaluation of mucosal and transmural lesions and this technique is thought to be necessary to understand the past and present disease status. Double balloon enteroscopy should be reserved for specific situations in which biopsy samples from suspected involved areas are important for diagnosis or in which a dilatation of stricture is reasonable.

Background Host-intestinal microbial interaction plays an important role in the pathogenesis of inflammatory bowel diseases (IBDs). The surface molecules of the intestinal epithelium act as receptors for bacterial adhesion and regulate the intestinal bacteria. Some known receptors are the mucosal blood type antigens, which are regulated by the fucosyltransferase2 (FUT2) gene, and individuals who express these antigens in the gastrointestinal tract are called secretors. Recent research has revealed that the FUT2 gene is associated with Crohn's disease (CD) in western populations. Methods To clarify the contribution of mucosal blood type antigens in IBD, we determined the incidence of five previously reported single-nucleotide polymorphisms of the FUT2 gene in Japanese patients. We also used immunohistochemistry to investigate the antigen expression in mucosal specimens from IBD patients and animal models. Results Genetic analysis revealed that all of the patients with colonic CD were secretors, whereas the incidence of secretors was 80, 80, 67, and 80%, respectively, for the control, ileocolonic CD, ileal CD, and ulcerative colitis groups (P = 0.036). Abnormal expression of blood type antigens was observed only in colonic CD. Interleukin- 10 -/- mice, but not dextran sulfate sodium colitis mice, had enhanced colonic expression of blood type antigens, and the expression of these antigens preceded the development of colitis in the interleukin-10 -/- mice. Conclusions FUT2 secretor status was associated with colonic-type CD. This finding, taken together with the immunohistochemistry data, suggests that the abnormal expression of blood type antigens in the colon may be a unique and essential factor for colonic CD. © Springer 2011.

Interleukin (IL)-12 is a key factor that induces T helper cell type 1-mediated immunity and inflammatory diseases. In some colitis models, such as IL-10 knock-out (KO) mice, IL-12 triggers intestinal inflammation. An abundant amount of IL-12 is produced by intestinal macrophages in response to stimulation by commensal bacteria in IL-10 KO mice. Intact bacteria are more potent inducers of macrophage IL-12 production than cell surface components in this model. This suggested that cell surface receptor signalling and intracellular pathogen recognition mechanisms are important for the induction of IL-12. We addressed the importance of intracellular recognition mechanisms and demonstrated that signal transducers and activator of transcription 1 (STAT1) signalling activated bacterial phagocytosis and was involved in the induction of abnormal IL-12 production. In IL-10 KO mouse bone marrow-derived (BM) macrophages, Escherichia coli stimulation induced increased IL-12p70 production compared to lipopolysaccharide combined with interferon (IFN)-γ treatment. Significant repression of IL-12 production was achieved by inhibition of phagocytosis with cytochalasin D, and inhibition of de novo protein synthesis with cycloheximide. Induction of IFN regulatory factors-1 and -8, downstream molecules of STAT1 and the key transcription factors for IK-12 transcription, following E. coli stimulation, were mediated by phagocytosis. Interestingly, STAT1 was activated after stimulation with E. coli in IL-10 KO BM macrophages, although IFN-γ could not be detected. These data suggest that molecules other than IFN-γ are involved in hyper-production mechanisms of IL-12 induced by E. coli stimulation. In conclusion, enteric bacteria stimulate excessive IL-12p70 production in IL-10 KO BM macrophages via phagocytosis-dependent signalling. © 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.

We have previously demonstrated that IL-7 is essential for the persistence of colitis as a survival factor of colitogenic IL-7Rα-expressing memory CD4+ T cells. Because IL-7Rα is broadly expressed on various immune cells, it is possible that the persistence of colitogenic CD4+ T cells is affected by other IL-7Rα-expressing non-T cells. To test this hypothesis, we conducted two adoptive transfer colitis experiments using IL-7Rα-/- CD4+CD25- donor cells and IL-7Rα-/- x RAG-2-/- recipient mice, respectively. First, IL-7Rα expression on colitic lamina propria (LP) CD4+ T cells was significantly higher than on normal LP CD4+ T cells, whereas expression on other colitic LP immune cells, (e.g., NK cells, macrophages, myeloid dendritic cells) was conversely lower than that of paired LP cells in normal mice, resulting in predominantly higher expression of IL-7Rα on colitogenic LP CD4+ cells, which allows them to exclusively use IL-7. Furthermore, RAG-2-/- mice transferred with IL-7Rα-/- CD4+CD25- T cells did not develop colitis, although LP CD4+ T cells from mice transferred with IL-7Rα-/- CD4+CD25- T cells were differentiated to CD4+ CD44highCD62L- effector-memory T cells. Finally, IL-7Rα-/- x RAG-2 -/- mice transferred with CD4+CD25- T cells developed colitis similar to RAG-2-/- mice transferred with CD4 +CD25- T cells. These results suggest that IL-7Rα expression on colitogenic CD4+ T cells, but not on other cells, is essential for the development of chronic colitis. Therefore, therapeutic approaches targeting the IL-7/IL-7R signaling pathway in colitogenic CD4 + T cells may be feasible for the treatment of inflammatory bowel diseases. Copyright©2011 by The American Association of Immunologists, Inc.

Th17 cells and Th1 cells coordinate to play a critical role in the formation of inflammatory bowel diseases. To examine how Th17 and Th1 cells are regulated at inflammatory sites, we used Th1-dominant CD4+CD45RB high T cell-transferred RAG-2-/- and Th1/Th17-mixed IL-10-/- mice. Interestingly, not only did colitic RAG-2 -/- mice that were parabiosed with WT mice show significant amelioration of colitis, but amelioration of disease was also observed in those parabiosed with colitic IL-10-/- mice. To assess the interference between Th1 and Th17 colitogenic T cells, we co-transferred colitogenic CD4 + T cells from the lamina propria (LP) of CD4+CD45RB high T cell-transferred RAG-2-/- mice and IL-10 -/- mice into RAG-2-/- mice. Surprisingly, the co-transferred RAG-2-/- mice showed a vast cellular infiltration of LP CD4+ T cells similar to that seen in RAG-2-/- mice re-transferred with the cells from colitic RAG-2-/- mice alone, but the co-transferred RAG-2-/- mice did not have the wasting symptoms, which are also absent in RAG-2-/- mice transferred with cells from colitic IL-10-/- mice alone. Furthermore, the percentages of Th1 and Th17 cells originating from IL-10-/- mice and those of Th1 cells originating from colitic RAG-2-/- mice were all significantly decreased in the co-transferred mice as compared with the singly-transferred paired RAG-2-/- mice, suggesting that Th1 and Th17 cells are in competition, and that their orchestration results in a merged clinical phenotype of the two types of murine colitis. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

Here, we report an adult patient with IgA-related enteropathy complicated with massive intestinal bleeding and acute renal failure, but without skin lesions. Surgical resection of the small intestine and steroid pulse therapy was performed. Histopathology revealed significant deposition of IgA and C3 in the small vessels of the intestine and the kidney mesangium. Although skin purpura was absent, the histopathology and clinical manifestations suggested that the pathophysiology was similar to Henoch-Schönlein purpura (HSP), implying IgA-related enteropathy as a subclass of HSP. Retrospective analysis indicates that terminal ileum lesions may be a poor prognostic indicator. © 2010 The Japanese Society of Internal Medicine.

IL-2 and IL-7 share a common γ-chain receptor and are critical for T-cell homeostasis. We aimed to clarify the reciprocal roles of IL-2 and IL-7 in the development and persistence of chronic colitis. We performed a series of adoptive transfers of IL-2-/- CD4+CD45RBhigh T cells into RAG-2-/- mice and assessed the role of IL-2 in the induction of IL-7Rα on colitogenic CD4+ T cells and the development of chronic colitis. RAG-2-/- mice transferred with WT but not with IL-2-/- CD4+CD45RBhigh T cells developed Th1/Th17-mediated colitis. Consistently, re-expression of IL-7Rα was severely impaired on IL-2-/- but not on WT CD4+ T cells from the transferred mice. To exclude a contribution of the preclinical autoimmunity of IL-2-/-mice, WT Ly5.1+ or IL-2 -/- Ly5.2+ CD4+CD45RBhigh T cells from GFP mice previously transplanted with the same number of WT and IL-2 -/- BM cells were transferred into RAG-2-/- mice. RAG-2-/- mice transferred with IL-2-/--derived CD4 + CD45RBhigh T cells did not develop colitis, but their splenic CD4+ T cells changed from effector-memory to central-memory type. These results show that IL-2 is critically involved in the establishment and maintenance of IL-7-dependent colitogenic memory CD4+IL- 7Rαhigh T cells. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.