Published papers

The etiology and pathogenesis of inflammatory bowel disease remains an area under intense investigation. Crohn's disease (CD) is characterized by a marked accumulation of activated Th1 type CD4+ T cells and macrophages in inflamed intestinal mucosa. IL-18 is a recently described cytokine that mainly exists in activated macrophages and shares biological activities with IL-12 in driving the development of Th1 type CD4+ T cells by inducing interferon-γ. To clarify the role of IL-18 in intestinal inflammation in CD, we assessed the functional role of IL-18 in regulating intestinal mucosal lymphocytes in human CD and murine CD model.

Disorders in enteric bacteria recognition by intestinal macrophages (Mφ) are strongly correlated with the pathogenesis of chronic colitis; however the precise mechanisms remain unclear. The aim of the current study was to elucidate the roles of Mφ in intestinal inflammation by using an IL-10-deficient (IL-10-/-) mouse colitis model. GM-CSF-induced bone marrow-derived Mφ (GM-Mφ) and M-CSF-induced bone marrow-derived Mφ (M-Mφ) were generated from bone marrow CD11b+ cells. M-Mφ from IL-10-/- mice produced abnormally large amounts of IL-12 and IL-23 upon stimulation with heat-killed whole bacteria Ags, whereas M-Mφ from wild-type (WT) mice produced large amounts of IL-10 but not IL-12 or IL-23. In contrast, IL-12 production by GM-Mφ was not significantly different between WT and IL-10-/- mice. In ex vivo experiments, cytokine production ability of colonic lamina propria Mφ (CLPMφ) but not splenic Mφ from WT mice was similar to that of M-Mφ, and CLPMφ but not splenic Mφ from IL-10-/- mice also showed abnormal IL-12p70 hyperproduction upon stimulation with bacteria. Surprisingly, the abnormal IL-12p70 hyperproduction from IL-10-/- from IL-10-/- mice was improved by IL-10 supplementation during the differentiation process. These results suggest that CLPMφ and M-Mφ act as anti-inflammatory Mφ and suppress excess inflammation induced by bacteria in WT mice. In IL-10 -/- mice, however, such Mφ subsets differentiated into an abnormal phenotype under an IL-10-deficient environment, and bacteria recognition by abnormally differentiated subsets of intestinal Mφ may lead to Th1-dominant colitis via IL-12 and IL-23 hyperproduction. Our data provide new insights into the intestinal Mφ to gut flora relationship in the development of colitis in IL-10-/- mice.

Background and aims: The pathogenesis of Crohn's disease (CD), a chronic inflammatory bowel disease characterised by a Th1 immune response, remains unclear. Osteopontin (OPN) is a phosphoprotein known as an adhesive bone matrix protein. Recent studies have shown that OPN plays an important role in lymphocyte migration, granuloma formation, and interleukin 12 (IL-12) production. The present study investigated expression and the pathophysiological role of OPN in CD. Methods: Plasma OPN concentration was measured by enzyme linked immunosorbent assay. Expression of OPN in human intestinal mucosa was determined using reverse transcription-polymerase chain reaction and western blot, and localisation of OPN was examined by immunohistochemistry. Expression of integrin s, an OPN receptor, on lamina propria mononuclear cells (LPMC) was assessed by flow cytometry. Functional activation of OPN in LPMC was investigated by measuring the production of cytokines. Results: Plasma OPN concentration was significantly higher in patients with CD compared with normal controls or patients with ulcerative colitis (UC). OPN was upregulated in intestinal mucosa from UC and CD patients. OPN producing cells were epithelial or IgG producing plasma cells, or partial macrophages. OPN was detected in areas surrounding granuloma from mucosa in CD. Integrin βs expressing macrophages infiltrated inflamed mucosa in UC and CD; in contrast, there was no expression of integrin βs on intestinal macrophages in normal mucosa. OPN induced production of IL-12 from LPMC in CD but not in normal controls or UC. Conclusions: Increased OPN expression facilitates cytokine production and is closely involved in the Th1 immune response associated with CD.

Background: Nonpathogenic Escherichia coli strain Nissle1917 has been used as a probiotics in human inflammatory bowel disease; however, there are few reports examining its therapeutic effect on animal colitis models, and its therapeutic mechanisms remain unknown. The aim of this study was to elucidate the therapeutic effect and mechanism of Nissle1917 using murine acute and chronic colitis models. Methods: Two models were used. (1) Acute model: colitis was induced by administration of 1.3% dextran sodium sulfate for 7 days. Nissle1917 or phosphate-buffered saline were orally administered for 10 days. Mice were killed at day 10, and the colonic lesions were assessed macro- and microscopically. (2) Chronic model: IL-10-/- mice were treated with Nissle1917 or phosphate-buffered saline for 8 weeks. After 8 weeks of treatment, mice were killed to assess the colonic lesions macro- and microscopically. In the acute dextran sodium sulfate colitis model, viable, heat-killed, or genomic DNA of Nissle1917 was orally administered for 10 days, and the therapeutic effect was assessed. Results: In the acute model, Nissle1917 ameliorated body weight loss, disease activity index, and macro- and microscopic damage. In the chronic model, it also suppressed the mucosal inflammatory findings and histologic damages. Moreover, heat-killed Nissle1917 or its genomic DNA alone also ameliorated the acute DSS colitis and viable bacteria macro- and microscopically. Conclusions: Nonpathogenic E. coli strain Nissle1917 prevents both acute and chronic colitis, and its anti-inflammatory effect is exhibited not only by viable bacteria but also by heat-killed bacteria or its DNA.

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.

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.

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.

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.

Background & Aims: Autoimmune responses against colonic epithelium may play a role in the development of colonic inflammation associated with ulcerative colitis (UC). In this study, we established and characterized B-cell lines and clones that produced anticolon antibody from inflamed colonic mucosa of UC subjects. Methods: B-cell lines were generated through Epstein-Barr virus transformation of lamina propria lymphocytes (LPLs) from colonic mucosa and peripheral blood lymphocytes, and these lines were screened for the production of anticolon antibodies. B-cell lines were then cloned by limiting dilution culture, and messenger RNA expression of immunoglobulin heavy-chain variable region (VH) was assessed. Results: VH gene families used in B-cell lines established from LPLs of normal controls were diverse, and B-cell lines from UC LPLs expressed a restricted VH3 family usage. All 15 clones from UC used a restricted VH3 gene family, whereas diverse VH gene families were used by 24 clones from normal controls. The analysis of nucleotide sequences indicated that these clones were derived from various germline gene segments. Conclusions: The restricted VH gene usage in anticolon autoantibodies producing B-cell clones suggests that a particular antigenic stimulus contributes to the pathogenesis of UC.