Role of intestinal subepithelial myofibroblasts in inflammation and regenerative response in the gut

Pharmacology & Therapeutics 114 (2007) 94 – 106 www.elsevier.com/locate/pharmthera

Associate editor: J.S. Fedan

Role of intestinal subepithelial myofibroblasts in inflammation and regenerative response in the gut Akira Andoh a,⁎, Shigeki Bamba a , Mairi Brittan b , Yoshihide Fujiyama a , Nicholas A. Wright b a

Department of Gastroenterology, Shiga University of Medical Science, Seta-Tsukinowa, Otsu 520-2192, Japan b Queen Mary's School of Medicine and Dentistry, London, United Kingdom

Abstract Inflammatory bowel disease (IBD) is characterized by an ongoing mucosal inflammation caused by a dysfunctional host immune response to commensal microbiota and dietary factors. In the pathophysiology of IBD, mesenchymal cells such as intestinal subepithelial myofibroblasts (ISEMF) affect the recruitment, retention and activation of immune cells. Mesenchymal cells also promote resolution of inflammatory activity accompanied with balanced repair processes. The transient appearance of mesenchymal cells is a feature of normal wound healing, but the persistence of these cells is associated with tissue fibrosis. Recent studies suggest that mesenchymal cells derived from bone marrow (BM) stem cells play a crucial role in intestinal repair and fibrosis. This article focuses on recent knowledge about ISEMF in the field of immune response inflammation and repair. Two major topics were documented: interaction between interleukin (IL)-17-secreting CD4+ cells (Th-17 cells) and about role of BM-derived stem cells in mucosal regenerative response via differentiation to ISEMF. Recent therapeutic strategies targeting BM stem cells for IBD patients were also documented. © 2007 Elsevier Inc. All rights reserved. Keywords: Inflammatory bowel disease; Interleukin-17; Stem cells; Niche; Plasticity

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characterization of intestinal subepithelial myofibroblasts . . . . . . . . . . . . . . Extracellular matrix turnover and matrix metalloproteinases secretion . . . . . . . . Interleukin-17 and intestinal subepithelial myofibroblasts. . . . . . . . . . . . . . . 4.1. Interleukin-17-producing T cells are distinct lineage from Th1 and Th2 cells . 4.2. Expression of IL-23 p19 subunit by ISEMF . . . . . . . . . . . . . . . . . . 4.3. Modulation of intestinal subepithelial myofibroblast functions by IL-17 . . . 4.4. Interaction between IL-17 and IL-22. . . . . . . . . . . . . . . . . . . . . . Other factors expressed by intestinal subepithelial myofibroblasts . . . . . . . . . . 5.1. Stem cell factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Vascular endothelial growth factor . . . . . . . . . . . . . . . . . . . . . . . 5.3. IL-11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The gastrointestinal stem cells and the stem cell “niche” . . . . . . . . . . . . . . . The Wnt/β-catenin signaling pathway and intestinal subepithelial myofibroblasts . . Plasticity of adult bone marrow cells . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Adult bone marrow cells to gut epithelial cells . . . . . . . . . . . . . . . . 8.2. Bone marrow contribution to intestinal subepithelial myofibroblasts . . . . .

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⁎ Corresponding author. Department of Internal Medicine, Shiga University of Medical Science, Seta-Tukinowa, Otsu 520-2192, Japan. Tel.: +81 77 548 2217; fax: +81 77 548 2219. E-mail address: [email protected] (A. Andoh). 0163-7258/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2006.12.004

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Therapeutic approaches targeting induction of bowel disease. . . . . . . . . . . . . . . . . 10. Conclusion . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

1. Introduction Ulcerative colitis (UC) and Crohn's disease (CD) comprise the 2 most common forms of inflammatory bowel disease (IBD) and are characterized by an ongoing mucosal inflammation caused by a dysfunctional host immune response to commensal microbiota and dietary factors (Riddle, 1995; Stenson, 1995; Podolsky, 2002; Hibi & Ogata, 2006). Intestinal inflammation has traditionally been considered a process, in which effector immune cells cause the destruction of other mucosal cells that behave as passive bystander targets (Fiocchi, 1997). The chronic inflammatory process leads to destruction of the epithelial barrier and subsequent epithelial ulceration, which permits easy access of luminal microbiota and dietary antigens to the cell resident in the lamina propria. In this response, mesenchymal cells such as stromal fibroblasts and myofibroblasts affect the recruitment, retention and activation of immune cells, through their synthesis of cytokines, chemokines, eicosanoids and extracellular matrix (ECM) components (Powell et al., 1999a, b; Andoh et al., 2005a,b). The importance of mesenchymal cells to the perpetuation of chronic inflammation has been previously appreciated in the literature (Fiocchi, 1997; Andoh et al., 2002b). Resolution of inflammatory activity is associated with balanced repair processes that facilitate tissue remodeling and, in turn, restore normal intestinal architecture and mucosal structure. The transient appearance of mesenchymal cells is a feature of normal wound healing, but the persistence of these

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marrow-derived stem cells in inflammatory . . . . . . . . . . . . . . . . . . . . . . . 103 . . . . . . . . . . . . . . . . . . . . . . . 104 . . . . . . . . . . . . . . . . . . . . . . . 104

cells is associated with excessive collagen deposition and fibrosis, which is frequently observed in CD patients and involves mesenchymal cell hyperplasia, tissue disorganization and fibrillar collagen deposition (Riddle, 1995; Stenson, 1995). Recent studies suggest that mesenchymal cells derived from bone marrow (BM) stem cells play a crucial role in intestinal repair and fibrosis (Pucilowska et al., 2000; Brittan & Wright, 2002, 2004). This article focuses on recent knowledge about intestinal subepithelial myofibroblasts (ISEMF) in the field of immune response, inflammation and repair. 2. Characterization of intestinal subepithelial myofibroblasts The subepithelial mesenchymal cells and their secreted basement membrane factors comprise the lamina propria, which provides a supporting network for the epithelial cells and regulates epithelial cell function (Powell et al., 1999a,b; Andoh et al., 2005a,b). The lamina propria contains 2 intestinal myofibroblast populations: the interstitial cells of Cajal (ICC) and ISEMF. ICC are pacemaker cells located in an intramuscular space between the submucosa and muscularis propria, which regulate gastrointestinal smooth muscle motility, facilitate the propagation of electrical events and regulate neurotransmission (Sanders et al., 2002). ISEMF, also called pericryptal fibroblasts, are a syncytium of α-smooth muscle actin (α-SMA)-positive stromal

Fig. 1. Colonic subepithelial myofibroblasts (ISEMF). (A and B) Immunostaining for α-SMA in the human colon. (C) Isolated human colonic ISEMF. (D) Immunostaining for α-SMA in isolated ISEMF.

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(mesenchymal) cells, which reside subjacent to the basement membrane of the small and large intestines (Powell et al., 1999a,b; Andoh et al., 2005a,b; Fig. 1). These cells are members of a family of functionally related cells, including hepatic Ito cells, glomerular mesangial cells, and orbital and synovial fibroblasts (Valentich et al., 1997). In the region of the crypts, ISEMF are scaphoid in appearance and overlap like shingles on a roof (Kaye et al., 1968; Powell et al., 1999b). ISEMF form a vascular, fenestrated sheath subjacent to the epithelial cells, innervated by the enteric nervous system, and connected in a cellular syncytium via gap and adherens junctions. In the upper regions of the crypts and the small intestinal villi, the ISEMF display a stellate morphology (Joyce et al., 1987; Komuro & Hashimoto, 1990). ISEMF have a broad range of functions, including regulation of epithelial cell proliferation and differentiation, mucosal protection and wound healing, water and electrolyte transport, and ECM metabolism affecting the growth of the basement membrane (Powell et al., 1999a,b; Andoh et al., 2005a,b). ISEMF are specialized mesenchymal cells that exhibit the ultrastructural features of both fibroblasts and smooth muscle cells and can be characterized by positive immunoreactivity for α-SMA and vimentin but are negative for desmin (Sappino et al., 1990; Mahida et al., 1997; Powell et al., 1999a,b). ISEMF are distinguished from smooth muscle cells, which express αSMA but are negative for vimentin. Platelet-derived growth factor (PDGF-BB), basic fibroblast growth factor (bFGF) and insulin-like growth factor (IGF-I) significantly increased the uptake of tritiated [3H]thymidine into isolated human ISEMF (Okuno et al., 2002). In particular, PDGF had a strong stimulatory effect, although interleukin (IL)1β, TNF-α, epidermal growth factor (EGF), keratinocyte growth factor (KGF) and TGF-β showed no effects (Okuno et al., 2002). In contrast, Jobson et al. (1998) showed growth stimulatory effects of IL-1β, TNF-α and EGF on ISEMF, as well as PDGF, bFGF and IGF-I. In IBD mucosa the number of α-SMA-positive cells is increased as compared to the normal mucosa (Riddle, 1995; Pucilowska et al., 2000; Andoh et al., 2002b). McKaig et al. (2002) demonstrated that proliferation of ISEMF isolated from CD patients was significantly greater than that of ISEMF derived from normal or UC patients. Pucilowska et al. (2000) showed that IGF-I and procollagen α1(I) mRNA showed an overlapping distribution within fibrotic mucosa with growth of fibroblasts and/or myofibroblasts (Pucilowska et al., 2000). 3. Extracellular matrix turnover and matrix metalloproteinases secretion The ECM consists of collagens, other glycoproteins, and proteoglycans (Scott-Burden et al., 1989; Simon-Assmann et al., 1995). ISEMF constitutively secrete large amounts of ECM factors modulated by proinflammatory cytokines and growth factors. In isolated ISEMF, the secretion of types I, IV collagens and fibronectin into the culture medium was stimulated by IL-1β, TNF-α and TGF-β (Okuno et al., 2002; Simmons et al., 2002).

Matrix metalloproteinases (MMP) belong to the major enzyme group capable of degrading ECM and basement membrane components (Nagase & Woessner, 1999; Nelson et al., 2000). MMP-1 is an interstitial collagenase and causes the degradation of collagen types I, II and III. MMP-2 is termed gelatinase-A and degrades nonfibrillar collagen types IV and V. MMP-3 (stromelysin-1) is capable of degrading proteoglycans, laminin, fibronectin and nonfibrillar collages. The activity of MMP is controlled by specific tissue inhibitors of metalloproteinases (TIMP) and nonspecific inhibitors, such as β2macroglobulin (Nagase & Woessner, 1999; Nelson et al., 2000). A fine balance between MMP and TIMP controls the rate of ECM turnover under normal physiological conditions and in tissue remodeling during inflammation and wound healing. Previous studies showed that expression of MMP and TIMP are elevated in inflamed mucosa of IBD patients (Louis et al., 2000; von Lampe et al., 2000; McKaig et al., 2003). Unstimulated ISEMF expressed MMP-2 and TIMP-2 mRNA, but the mRNA expression for MMP-1, MMP-3 and TIMP-1 was weak (Yasui et al., 2004). IL-1β and TNF-α induced a marked increase in MMP-1, MMP-3 and TIMP-1 mRNA expression in intestinal ISEMF, but these factors did not alter basal MMP-2 and TIMP-2 mRNA expression. FGF-2 (bFGF) also stimulated MMP-1, MMP-3 and TIMP-1 mRNA expression (Yasui et al., 2004). Furthermore, we found that IL17, an activated T cell-derived cytokine, stimulates MMP-3 secretion in ISEMF (Bamba et al., 2003a). The basal secretion of large amounts of MMP-2, accompanied by the secretion of ECM, suggests that ISEMF actively participate in ECM metabolism, including basement membrane turnover. These observations indicate that proinflammatory cytokines and growth factors control MMP and TIMP expression in ISEMF, and that these are major factors contributing to the tissue remodeling process. 4. Interleukin-17 and intestinal subepithelial myofibroblasts 4.1. Interleukin-17-producing T cells are distinct lineage from Th1 and Th2 cells IL-17 was originally identified as cytotoxic T lymphocyteassociated antigen-8 and viral IL-17, the open reading frame 13 of T-lymphotropic Herpesvirus saimir (Kolls & Linden, 2004). Human IL-17 is a ∼ 20-kDa glycoprotein of 155 amino acids, with close sequence homology to both murine and viral IL-17 (Kolls & Linden, 2004). IL-17 secretion is limited to T lymphocytes, predominantly in memory CD45RO+ cells. In contrast, the IL-17 receptor is widely distributed in various cell types. Expression of IL-17 has been linked to a growing list of autoimmune and inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, Helicobacter pyloriassociated gastritis, bronchial asthma and renal allograft rejection (Cua et al., 2003; Kolls & Linden, 2004; Wynn, 2005). IL-17 is believed to contribute to the pathogenesis of these diseases by acting as a potent proinflammatory mediator (Kolls & Linden, 2004).

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Studies aimed at characterization of the IL-17-producing T cells (Th-17 cells) as products of a developmental lineage distinct from that of Th1 and Th2 T cells are emerging, with several reports that IL-23-stimulated CD4+ Th1 cells are the main source of IL-17 (Langrish et al., 2004, 2005; Fig. 2), although the molecular mechanisms controlling the development of Th-17 cells have remained unclear. Both IL-23 and IL12 use the IL-12p40 chain and interact with receptors that share the common IL-12 β1 subunit (IL-12Rβ1; Parham et al., 2002), and both Th1, positive for interferon (IFN)-γ, and Th-17 CD4+ T cells often coexist in inflamed tissues (Oppmann et al., 2000). Based on these observations, it has been widely believed that Th1 and Th-17 cells arise from a common Th1 precursor (Wynn, 2005), whereas intracellular cytokine staining studies demonstrated that there is little or no overlap between IL-17 and IFN-γ expression. Two recent studies support a hypothesis wherein Th-17 cells arise as a separate Th lineage from Th1 and Th2 T cells (Harrington et al., 2005; Park et al., 2005) and predict that they do not represent a population of Th1 cells that undergo further differentiation (Park et al., 2005; Wynn, 2005; Reinhardt et al., 2006). These studies demonstrated that IL-23 can induce the differentiation of naïve T cells into Th-17 cells through a mechanism that differs from the signals driving the development of Th1 cells (dependent on the transcription factors T-bet, STAT4 and STAT1) and Th2 cells (dependent on GATA-3 and STAT6; Fig. 2), indicating that Th-17 cells act as a separate and early lineage of effector T cells. When naive CD4+ T cells were cultured in the presence of anti-IFN-γ, predominant growth of Th-17 cells was induced. Unexpectedly, blockade of IL-4 also showed a stimulatory effect on Th-17 development. The combination with IL-23 together with anti-IL-4 and anti-IFNγ was the most potent cocktail for generation of Th-17 cells.

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Thus, the differentiation of Th-17 cells is most efficiently induced when Th1 and Th2 effector functions are simultaneously inhibited. Very recent studies showed a central role for TGF-β to initiate commitment of naive CD4+ T cells to Th-17 cells (Mangan et al., 2006). TGF-β induced Th-17 development in the absence of IL-23. Mice deficient in TGF-β1 are deficient in Th-17 cells and in circulating levels of IL-17. Furthermore, natural regulatory T cells (Tregs) could be substituted for the actions of TGF-β (Veldhoen et al., 2006). Other recent studies showed that IL-27 is one of the major inhibitory factors for Th17 development. Thus, these recent reports suggest that antagonistic cytokines are acting early to establish Th-17 lineage commitment. 4.2. Expression of IL-23 p19 subunit by ISEMF As mentioned above, IL-23 is a critical factor for induction of Th-17 cells. IL23 consists of p19 and p40 subunits. p19, a molecule structurally related to IL-6, G-CSF and the p35 subunit of IL-12, was recently discovered by searching databases with a computationally derived profile of IL-6. The major cellular source of the p19/p40 pair (IL-23) are the activated antigen-presenting cells, such as dendritic cells (DC) and/or macrophages (Belladonna et al., 2002; Oppmann et al., 2000). IL-23 induces the proliferation of memory T cells and stimulates IL-17 secretion (Oppmann et al., 2000; Belladonna et al., 2002). Furthermore, IL-23 can activate macrophages to produce IL-1β and TNF-α (Cua et al., 2003). Recently, the transgenic expression of the IL-23 subunit p19 was shown to result in systemic inflammation accompanied by elevated levels of serum IL-1β and TNF-α (Yang et al., 2000). We have recently shown that the IL-23 p19 subunit is inducible in colonic SEMF in response to IL-1β and TNF-α

Fig. 2. Schema of Th-17 cells differentiation. IL-23 induces the differentiation of naive T cells into Th-17 cells through a mechanism that differs from the signals driving the development of Th1 cells (dependent on the transcription factors T-bet, STAT4 and STAT1) and Th2 cells (dependent on GATA-3 and STAT6). Th-17 cells are a separate and early lineage of effector T cells. When naive CD4+ T cells were cultured in the presence of anti-IFN-γ, predominant growth of Th-17 cells was induced. Blockade of IL-4 also showed a stimulatory effect on Th-17 development. IL-27 also blocks Th-17 development.

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Fig. 3. Effects of various cytokines and growth factors on IL-23 p19 mRNA expression in human colonic ISEMF. The cells were stimulated for 12 hr, and IL23 p19 mRNA expression was determined by Northern blotting.

(Fig. 3). In particular, the effects of IL-1β were remarkable as compared to those of other cytokines. Furthermore, IL-1β and TNF-α synergistically induce IL-23 p19 mRNA expression (Zhang et al., 2005b; Sutton et al., 2006). Sutton et al. (2006) recently reported that IL-23 alone is a poor stimulus for T cell IL-17 production and that IL-1 is essential for its induction. IL-1 acted directly on T cells in synergy with IL23 to promote IL-17 secretion. TNF-α also synergized with IL-23 to induce IL-17, but this was dependent on IL-1. Combined with these findings, our observation in ISEMF suggests that a critical role of IL-1 in the induction of IL-17 secretion may be partially mediated by the induction of IL-23 secretion by IL-1. 4.3. Modulation of intestinal subepithelial myofibroblast functions by IL-17 The number of Th-17 cells was increased in the inflamed mucosa of IBD patients (Fujino et al., 2003). IL-17 expression was not detected in colonic mucosa from normal individuals, infectious colitis and ischaemic colitis. In the inflamed mucosa of active UC and CD patients, IL-17 protein and mRNA expression was clearly detectable in CD3+ T cells (Fujino et al., 2003). Furthermore, serum IL-17 levels were also significantly elevated in IBD patients (Fujino et al., 2003). This suggests a role of IL-17 in the pathophysiology of IBD. To investigate altered genes in response to IL-17 stimulus, we performed a cDNA microarray analysis in ISEMF isolated from human colon. As shown in Table 1, IL-17 upregulated several genes, which have been reported to exert proinflammatory actions in the pathophysiology of acute and/or chronic inflammation.

IL-17 stimulates IL-6, IL-8 and MCP-1 secretion via NF-κB and MAP kinase activation in ISEMF (Hata et al., 2002). IL-17 induced a rapid NF-κB activation after stimulation, and a blockade of NF-κB cascade markedly reduced these responses (Hata et al., 2002). Inhibition of MAPK significantly reduced the IL-17-induced increase in IL-6 and chemokine secretion. The combination of either IL-17 plus IL-1β or IL-17 plus TNFα enhanced cytokine secretion (Hata et al., 2002); in particular, the effects of IL-17 plus TNF-α on IL-6 secretion were much stronger than the other responses. This was dependent on the induction of IL-6 mRNA stability. As evidence obtained in studies of experimental animals, further supported by data from humans, suggests that excessive production of IL-6 and chemokines is involved in the pathogenesis of IBD and interaction between IL-17 and ISEMF might be the key in the pathophysiology of gut inflammation. A combination of IL-17 plus IL-4 synergistically enhanced IL-6 secretion in ISEMF (Andoh et al., 2002c). IL-17 also stimulates granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) in ISEMF (Andoh et al., 2005b). CSF regulate hematopoietic progenitor cell proliferation and enhance the functional activities of mature myeloid effector cells (Crosier & Clark, 1992). Both G-CSF and M-CSF are highly lineage specific in their action and, therefore, only support the proliferation of the progenitor cells of either granulocytes or macrophages, respectively (Crosier & Clark, 1992). Conversely, GM-CSF maintains both discrete progenitor cells that consist of macrophages or granulocytes. IL-17 weakly enhanced G-CSF release but did not affect GM-CSF and M-CSF release. IL-17 selectively enhanced TNF-α-induced G-CSF and GM-CSF release. The combination of IL-17 plus TNF-α induced a marked increase in NF-κB-DNA and AP-1-DNA binding activities. The adenovirus-mediated transfer of a stable form of IκB-α and/or a dominant negative mutant of c-Jun markedly

Table 1 Representative genes increased by IL-8 1 2 3 4 5 6 7 8 9 10 11 12 13

IL-6 (NM_00600.3569) (NM_00584.3576) Superoxide dismutase 2 (NM_00636.6648) CCL7 (NM_006273.6354) CCL2 (NM_002982.6347) CXCL1 (NM_001511.2911) CXCL2 (NM_002982.6347) CXCL3 (NM_002090.2921) CXCL6 (NM_002993.6372) Complement component 3 (NM_000064.718) MAP3K 8 (NM_005204.1326) Calpain small subunit 2 (NM_032330.84290) Carbonic anhydrase XII (NM_001218.771)

×4.6 ×8.9 ×4.3 ×2.8 ×2.38 ×26.1 ×9.8 ×11.7 ×12.5 ×5.6 ×3.3 ×3.7 ×4.4

ISEMF derived from the human colon were stimulated with IL-17 (200 ng/mL) for 12 hr, and the changes in gene expression were asseseed by InteliGene HS Human Expression Chip (Takara-Bio, Otsu, Japan). The fold change values were determined as a ratio of Cy5 signal intersity (IL-17 stimulated values)/Cy3 signal intensity (nonstimulated values). The data show the average of 3 independent analysis. The NCBI reference sequence code was presented following the gene name.

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inhibited the IL-17 plus TNF-α-induced G-CSF and GM-CSF mRNA expression. Furthermore, a stability study showed that IL-17 plus TNF-α markedly enhanced the stability of G-CSF and GM-CSF mRNA (Andoh et al., 2005b). As mentioned above, IL-17 induces proinflammatory gene expression in ISEMF. However, we found an antiinflammatory role of IL-17 in ISEMF. In ISEMF, TNF-α induces secretion of RANTES (regulated upon activation, normal T cell expressed and secreted), but IL-17 dose dependently inhibited the TNF-αinduced RANTES secretion (Andoh et al., 2002a). IL-17 significantly decreased the TNF-α-induced increase in RANTES promoter activity, and IL-17 actually blocked the TNF-α-induced RANTES gene transcription. The gel shift assay demonstrated that IL-17 did not modulate the TNF-αinduced NF-κB DNA-binding activity but markedly decreased TNF-α-induced IFN regulatory factor-1 (IRF-1) DNA-binding activity. Because cooperation between NF-κB and IRF-1 is important in TNF-α-induced RANTES gene expression, the major mechanism mediating the inhibitory effect of IL-17 may be achieved by the inhibition of IRF-1 DNA-binding activity (Andoh et al., 2002a). We investigated Toll-like receptor (TLR) expression in ISEMF (Zhang et al., 2005a). ISEMF isolated from human colon expressed mRNA for TLR1, TLR3–6 and TLR9, whereas expressions of mRNA for TLR2, TLR7, TLR8 and TLR10 were not detected. In contrast, Otte et al. (2003) demonstrated that isolated ISEMF express mRNA for TLR1–TLR9. These discrepancies may be due to differences in cell source. TLR4 is a receptor for lipopolysaccharide (LPS), and the expression of cyclooxigenase (COX)-2 protein and mRNA was rapidly induced by the addition of LPS and IL-17. Furthermore, LPS and IL-17 synergistically induced COX-2 mRNA and protein expression. Thus, COX-2 expression and prostaglandin synthesis might be induced by both T cell-derived factor (IL-17) and bacterial products (e.g., LPS) in the inflamed mucosa.

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combination of IL-17 plus IL-22 enhanced transcription factor activation, cytokine mRNA expression and protein secretion and, thus, cooperation of T-cell derived cytokines, such as IL-17 and IL-22, may be involved in the pathophysiology of IBD. 5. Other factors expressed by intestinal subepithelial myofibroblasts 5.1. Stem cell factor Stem cell factor (SCF) and its receptor c-kit have been extensively studied (Broudy et al., 1995; Liesveld et al., 1995). SCF has been shown to act synergistically with a number of cytokines to augument cellular proliferation, differentiation and/ or function. SCF increases detection of myeloid, erythroid and megakaryocytic progenitors in short-term and long-term marrow colony assays (Liesveld et al., 1995). SCF acts synergistically with erythropoietin, thrombopoietin (c-mpl ligand) and IL-11 to enhance murine megakaryocyte colony growth and increases megakaryocyte ploidy in vitro. Enhanced production of SCF is a potentially important response of the intestinal tract following exposure to cholera toxin or Salmonella typhimurium (Wang et al., 2000). SCF–c-kit interactions are important in the intestinal tract response to infection and this interaction may also regulate intraepithelial lymphocyte (IEL) function and proliferation during bacterial infections of the intestinal tracts (Wang et al., 2000). Isolated ISEMF constitutively secreted SCF into the supernatant (11.5 ± 6.2 ng/105 cells/ 24 hr). From the results of experiments of stimulation, various factors significantly increased basal secretion of SCF, for example, IL-17 (× 2.2), PDGF (× 2.4), KGF (× 2.6) and EGF (× 2.6), suggesting that SCF is constitutively produced by ISEMF in the human intestine and that ISEMF-derived SCF could play a physiologic role in maintaining IEL proliferation and function. Furthermore, ISEMF may regulate function and growth of ICC, which express c-kit.

4.4. Interaction between IL-17 and IL-22 5.2. Vascular endothelial growth factor IL-22 was originally described as an IL-9-induced gene and was termed “IL-10-related T cell-derived-inducible factor” (ILTIF) (Fickenscher et al., 2002; Wolk et al., 2004). IL-22 has 22% amino acid identity with IL-10 and belongs to a family of cytokines with limited homology to IL-10, namely IL-19, IL20, IL-22, IL-24 and IL-26. A major source of IL-22 is activated T cells, and IL-22 expression in other leukocyte populations, such as monocytes, DC, NK cells and neutrophils, is negligible. The polarization of T cells towards a Th1 phenotype further increases activation-induced IL-22 expression, whereas polarization towards a Th2 or regulatory phenotype reduces it. We recently found that IL-22 expressing cells are increased in inflamed mucosa of IBD patients (Andoh et al., 2005c). Furthermore, ISEMF are the primary expressing site for IL-22 receptor 1 (IL-22R1) in normal human colonic mucosa (Andoh et al., 2005c). In ISEMF, IL-22 upregulates the expression of inflammatory genes such as IL-6, IL-8, IL-11 and leukemia inhibitory factor (LIF) via NF-κB, AP-1 and MAP-kinase dependent pathways (Andoh et al., 2005c). Furthermore, the

Vascular endothelial growth factor (VEGF) is an angiogenic mitogen that specifically targets vascular endothelial cells, inducing vascular hyperpermeability in circulating macromolecules in several vascular beds, including those in the skin, subcutaneous tissues, peritoneal wall and mesentery (Fava et al., 1994; Koch et al., 1994). VEGF stimulates neoangiogenesis in regenerating tissue (Koch et al., 1994; Kanazawa et al., 2001). An abnormal microcirculatory system has also been implicated in the pathogenesis of IBD (Kanazawa et al., 2001); endothelin is significantly increased in patients with UC and CD (Griga et al., 1999; Kanazawa et al., 2001), and serum VEGF is elevated in both active UC and active CD patients but not in inactive patients (Griga et al., 1998; Kanazawa et al., 2001). ISEMF constitutively secreted VEGF (1.77 ± 0.1 ng/105 cells/ 24 hr), which was significantly enhanced by IL-1 (× 1.8), TNFα (× 1.6) and IL-1 plus IL-17 (× 2.4). Therefore, ISEMF-derived VEGF may be important for tissue repair via induction of angiogenesis in inflamed mucosa.

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5.3. IL-11 IL-11 is a member of the IL-6-type cytokine family that mediates its biologic activities via binding to a multimetric receptor complex that contains gp130 molecules (Schwertschlag et al., 1999). IL-11 was originally identified as a hematopoietic growth factor and stimulates multiple stages of megakaryocytopoiesis. IL-11 has demonstrated antiinflammatory properties in a variety of in vivo and in vitro models, and inhibits production of IL-1β, TNF-α and other proinflammatory cytokines from LPS-stimulated murine peritoneal macrophages. Furthermore, IL-11 reduces CD4+ T-cell production of Th1 cytokines, such as IL-12-induced IFN-γ, and enhances Th2 cytokines, such as IL-4 and IL-10. In the gastrointestinal tract, it is of particular interest that IL-11 prevents or improves the development of acute and chronic intestinal inflammation in animal models (Peterson et al., 1998; Bamba et al., 2003b). IL11 reversibly inhibited proliferation in untransformed small intestinal IEC18 cells, suggesting that IL-11 may be involved in the normal growth in the intestinal epithelium (Booth & Potten, 1995). The inhibitory response evoked by IL-11 is lost during carcinogenic transformation. We recently found that ISEMF secrete IL-11 in response to IL-1β and TGF-β (Bamba et al., 2003b). Both IL-1β and TGF-βı induced AP-1 activation, and a blockade of AP-1 activation markedly reduced the IL-1β- and TGF-βı-induced IL-11 mRNA expression. IL-1β and TGF-βı induced activation of ERK p42/44 and p38 MAP kinases, and MAP kinase inhibitors (SB202190, PD98059 and U0216) significantly reduced the IL-1β- and TGF-βı-induced IL-11 secretion. As mentioned above, IL-22 is a novel factor which stimulates IL-11 secretion (Andoh et al., 2005c), and IL-11R is expressed in intestinal epithelium (Kiessling et al., 2004). Thus, ISEMF-derived IL-11 may contribute to IBD pathophysiology as an anti-inflammatory factor. 6. The gastrointestinal stem cells and the stem cell “niche” Stem cells are undifferentiated primitive cells that exist within a tissue throughout the lifetime of an organism due to their ability to divide asymmetrically and undergo selfreplication and also to produce committed daughter cells that can differentiate to form all adult lineages within a tissue. The gastrointestinal epithelial stem cells are located and maintained within a mesenchymal niche, a specialized microenvironment that provides an optimal milieu for stem cell survival and function, believed to be created and maintained by the mesenchymal cells of the underlying subepithelial region, such as ISEMF. Although morphologically indistinct, adult epithelial stem cells can be defined functionally by this potential for asymmetrical division and are characterized by their residence within a stem cell compartment or “niche” situated toward the center of the gastric gland and near the base of the intestinal crypts. Mesenchymal–epithelial interactions between cells of the gastrointestinal mucosa and subjacent lamina propria are vital for the maintenance of normal epithelial cell function. For all adult stem cells, the “conceptual” stem cell niche should possess

3 standard constituents: the supporting cells and their secreted ECM to regulate stem cell behaviour via mesenchymal– epithelial crosstalk, the target cell range covered by the signaling molecules, and the stem cells themselves (Lin, 2002). Functionally, a niche is characterized by its persistence upon removal of the stem cells and, conversely, if stem cells are extracted from their niche, they cease to retain their stem cell potential, or “stemness,” and become committed to differentiation (Spradling et al., 2001). Niches can be colonized following transplantation of isolated stem cells (Brinster & Zimmermann, 1994) or the migration of cells from other niches (Oshima et al., 2001; Nishimura et al., 2002). The ability of niches to dynamically modulate cellular behaviour indicates a regulatory role in cellular transdifferentiation (Gussoni et al., 1999; Brazelton et al., 2000; Lagasse et al., 2000) and in proliferative disorders (Kim & Shibata, 2002). Kai and Spradling (2003) studied Drosophila ovarioles which maintain 2–3 germline stem cells in a niche requiring adhesive stromal cap cells. After experimentally emptying the germline stem cell niche, cap cell activity persisted for several weeks. Subsequently, follicle cell progenitors, including somatic stem cells, entered the niche and the cap cells provided support to these newly incorporated cells. In this context, it becomes a matter of great significance that, like cap cells, ISEMF influence epithelial cell proliferation and regeneration through epithelial–mesenchymal crosstalk and, ultimately, may determine epithelial cell fate. 7. The Wnt/β-catenin signaling pathway and intestinal subepithelial myofibroblasts The signaling protein Wnt, of which there are 19 family members identified in humans, activates the cytoplasmic phosphoprotein “dishevelled” through its receptor “frizzled,” causing inhibition of GSK3β and a resultant accumulation of cytosolic β-catenin (Kinzler & Vogelstein, 1996). β-catenin then translocates to the nucleus and interacts with members of the Tcf/LEF (T-cell factor/lymphocyte enhancer factor) family of DNA binding proteins, converting them from transcriptional repressors to activators and, hence, causing activation of downstream target genes which increase proliferation, including c-myc, tcf-1, cyclin D1, c-Jun, Fra-1, urokinase-type plasminogen activator receptor, fibronectin, CD44 and MMP (Brittan & Wright, 2002). Wnt signaling has distinct functions within the intestinal stem cell region. In the normal intestine, it is now known that the nuclear expression of β-catenin/TCF, which is upregulated by Wnt protein, is confined to the bottom of the normal crypts. Pinto et al. (2003) investigated the contribution of Wnt signaling to gastrointestinal epithelial proliferation using an adenovirus which mediated Dickkopf1 expression (Ad Dkk1), the soluble canonical Wnt inhibitor. Ad Dkk1 inhibited epithelial proliferation in the small intestine and colon, accompanied by progressive architectural degeneration with reduced number and size of crypts and villi. Although accumulating evidence suggests the importance of Wnt signaling in maintaining the stem cell “niche,” the source of the Wnt signaling and its regulation is still obscure. ISEMF are present immediately beneath

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the basement membrane, just under the epithelial cells and it is speculated that ISEMF form and maintain the stem cell niche. We have demonstrated the expressions of Wnt2, Wnt3, Wnt4, Wnt5a and Wnt5b mRNA in cultured murine ISEMF, whereas the expressions of Wnt3, Wnt5a and Wnt5b mRNA were also detected in crypt epithelium. In addition, frizzled (Fzd) mRNA (Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6 and Fzd7) were detected in murine ISEMF and crypt epithelium (Bamba et al., unpublished observation). Wnt mRNA expression was observed mainly in intestinal ISEMF; on the other hand, the expression of Fzd mRNA was observed in both intestinal ISEMF and crypt epithelium. The source of Wnt signaling is predominantly from ISEMF, consistent with a role of these cells in the maintenance of the stem cell niche. Fzd mRNA expression in ISEMF suggests that Wnt proteins secreted from ISEMF act not only in a paracrine manner but also in an autocrine manner. 8. Plasticity of adult bone marrow cells Adult stem cells from several tissues, in addition to their fundamental role of formation of all adult lineages within their native tissue, can extricate their niche and engraft within foreign tissues and transform to contribute to adult lineages within these tissues. This phenomenon is commonly termed “plasticity,” or “transdifferentiation.” Adult BM cells contribute to adult lineages within several nonhaematopoietic tissues, including the gastrointestinal tract (Brittan et al., 2002; Okamoto et al., 2002b), liver (Petersen et al., 1999; Alison et al., 2000; Lagasse et al., 2000; Theise et al., 2000), muscle (Ferrari et al., 1998), heart (Orlic et al., 2001), brain (Eglitis & Mezey, 1997) and kidney (Poulsom et al., 2001). 8.1. Adult bone marrow cells to gut epithelial cells The contribution of BM to the intestinal epithelium has been speculated for many years, although this notion was refuted by Cheng and Leblond (1974) using a [3H]thymidine-labeled BM transfusion model and in similar experiments using a genetic marker (Cairnie, 1976). The “Unitarian Theory” proposed by Cheng and Leblond (1974) has been widely accepted: that intestinal epithelial cells are provided by intestinal stem cells. However, there are several reports that BM cells can form both epithelial and mesenchymal lineages in the gut (Krause et al., 2001; Korbling et al., 2002; Okamoto et al., 2002b). Okamoto et al. (2002b) claimed that BM cells contribute to the human gastric and intestinal epithelium, whereas Korbling et al. (2002) also reported donor-derived cells in the gastric epithelium after peripheral blood stem cell transplantation. Moreover, Krause et al. (2001) demonstrated that a single BM-derived stem cell can engraft into intestinal epithelium in the mouse (Krause et al., 2001). In perhaps the most convincing report (Okamoto et al., 2002b), the same patient with graft-versus-host disease (GVHD) was observed at different time points, and a 10-fold to 15-fold increase in engrafted BM-derived cells in the small intestine was observed in the recovery phase. There was also a 40-fold to 50-fold increase of engrafted BM-derived epithelial

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cells in the regenerative epithelium of gastric ulcers among transplanted patients (Okamoto et al., 2002b). However, it is not clear whether the BM-derived cells were engrafted as shortlived epithelial cells or as long-lived epithelial cells that were still undifferentiated and multipotent, although the absence of clonally derived units would suggest that crypt stem cells are not formed by BM. It is important to note that donor-derived intestinal epithelial cells are single cells randomly interspersed throughout the crypts and villi, and the incidence of BMderived intestinal epithelial cells post-transplant are variable and are generally low, ranging from 0.04% (Spyridonidis et al., 2004) to ∼ 10% (Jiang et al., 2002). However, BM contribution to epithelial cells in human intestinal biopsies was increased between 5-fold and 50-fold in tissues damaged by GVHD, compared to intact epithelium (Okamoto et al., 2002a; Spyridonidis et al., 2004), although the low levels of engraftment and the lack of donor-derived cell clusters in crypts or villi indicate that it is unlikely that the BM cells form intestinal epithelial stem cells or undergo proliferation. We have performed many male-to-female BM engraftment experiments to try and show epithelial engraftment in normal, colitic and GVHD environments. In no case have we seen clonal engraftment in small or large intestines. Some morphologically “epithelial” Y-positive cells have turned out to be CD45positive T cells. We believe such cells to be a possible confounding issue in examining BM engraftment into the gut epithelium (C.-Y. Lee, unpublished observations). 8.2. Bone marrow contribution to intestinal subepithelial myofibroblasts We analyzed the colons and small intestines of female mice that had received a BM transplant from male donors, as well as gastrointestinal biopsies from female patients with GVHD after BM transplantation from male donors (Brittan et al., 2002). BM cells frequently engrafted into the mouse small intestine and colon, forming ISEMF within the lamina propria. In situ hybridisation confirmed the presence of a Y chromosome in these cells, and their positive immunostaining for α-SMA with negativity for desmin, the mouse macrophage marker F4/80 and the haematopoietic precursor marker CD34 determined their phenotype as pericryptal myofibroblasts. In a further study, mice were given a gender-mismatched BM transplant, and colitis was induced by an intracolonic injection of TNBS 6 weeks later. We observed a significant increase in BM contribution to colonic ISEMF in inflamed areas compared to the normal adjacent mucosa (Brittan et al., 2005), and, interestingly, foci of donor-derived colonic ISEMF were frequently observed in both the normal and inflamed mucosa. In addition, we have used an IL-10−/− chronic colitis model to assess the contribution of BM to colonic ISEMF in the inflamed mucosa. At 14 weeks post-transplantation, IL-10−/− female mice that had received IL-10−/− male BM had severe colitis and up to 45% of colonic ISEMF were of donor origin (Bamba et al., 2006) (Fig. 4). However, IL-10−/− mice receiving wild-type BM had no colitis, and ∼ 30% of colonic ISEMF were BM derived (Bamba et al., 2006). In both our models of colitis, BM engraftment was

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Fig. 4. Bone marrow-derived colonic ISEMF in IL-10−/− mice. The Y chromosome is detected as a brown dot in the cell nucleus. Red staining of the cytoplasm reflects positivity for α-SMA. After 14 weeks of bone marrow transplantation, IL-10−/−-to-IL-10−/− developed extensive colitis (A). BM-derived colonic ISEMF were frequently observed in these mice (B) (boxed area of A). Transverse sections of crypts show that they are surrounded by colonic ISEMF of BM origin (C).

enhanced in the inflamed colon, and BM-derived ISEMF were morphologically consistent with an “activated” phenotype, i.e., upregulated expression of chemokines, cytokines, growth factors and adhesion molecules in response to regenerative pressure (Powell et al., 1999b). This significant increase in BM cell engraftment and morphological features of an activated phenotype imply a functional role of BM in tissue regeneration. Importantly, BM contributed to ISEMF under normal circumstances (Fig. 5). However, previous reports of myofibroblast apoptosis after irradiation in the rodent and human gut (Wiernick & Perrins, 1975; Thiagarajah et al., 2000) indicate that BM engraftment into the pericryptal sheath in these models may be a result of the damage incurred by irradiation required prior to BM transplant. The origins and kinetics of myofibroblasts are yet to be fully understood. Myofibroblasts are thought to be derived from 2 major sources: BM (Brittan et al., 2002, 2005) and/or locally activated-fibroblasts in response to TGF-β1 (Fritsch et al., 1997) (Fig. 5). The kinetics of stromal myofibroblasts has been studied using [3H]thymidine pulse labeling in rabbit colon and adult mouse jejunum, suggesting that these cells, similar to intestinal epithelial cells, proliferate, migrate and differentiate along the crypt–villus axis (Pascal et al., 1968; Marsh & Trier, 1974). However, it should be noted that migration could not be confirmed in later studies with mouse small intestine and colon (Neal & Potten, 1981). Our observations of rows of BM-derived ISEMF in both the normal and inflamed mouse colon highlight the possibility that these cells may proliferate and, thus, give rise to differentiated myofibroblast progeny that possess a Y chromosome. Activated colonic ISEMF have strong proinflammatory effects (Rogler et al., 2001) and many in vitro experiments have revealed that colonic ISEMF have effector functions in the inflamed mucosa via secretion of proinflammatory cytokines and chemokines (Davidson et al., 2000;

Fig. 5. Derivation and role of ISEMF in the inflamed mucosa. Colonic ISEMF are thought to be (A) progeny of indigenously derived ISEMF, (B) of BM origin or (C) activated fibroblasts in response to TGF-β stimulation. BM-derived ISEMF were more frequently observed in the inflamed mucosa as compared with the normal mucosa. Crypt cell hyperplasia is frequently observed in the inflamed mucosa, and this is mediated by KGF secreted by TNF-α-stimulated ISEMF. Epithelial stem cells reside within a niche formed by a group of ISEMF at the bottom of the crypt. The expression of nuclear β-catenin, which is upregulated by Wnt protein, is confined to the bottom of the crypt. These suggest a crucial role for Wnt signaling in maintaining stem cell niche. Expression of Wnt mRNA is detectable in isolated ISEMF, suggesting that ISEMF are local source of Wnts.

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Bamba et al., 2003a). In this context, our results suggest that these “activated” donor-derived ISEMF may contribute to the flare and progress of colonic inflammation in IL-10−/− mice receiving IL-10−/− BM. 9. Therapeutic approaches targeting induction of bone marrow-derived stem cells in inflammatory bowel disease IBD patients are mainly treated with immunosuppressive drugs such as corticosteroid and immunosuppressant. However, based on the notion that BM-derived stem cells transdifferentiate into mucosal compartments, several therapeutic approaches that target induction of circulating BM-derived stem cells have been recently tried in IBD patients. Such approaches may contribute to mucosal repair through stimulation of regenerative reponses via differentiation of BM-derived stem cells. Significant amounts of hematopoietic stem cells are circulating in peripheral blood in active UC patients (Fig. 6). Leukocytapheresis therapy, a therapeutic strategy of extracorporeal immunomodulation for active UC patients, is effective in 70% of patients (Andoh et al., 2004; Sawada et al., 2005) and increases number of circulating hematopoietic stem cells by 2fold to 3-fold after treatment (A. Andoh, unpublished data). The mechanism underlying leukocytapheresis effects remains unclear, but removing activated leukocytes from the peripheral blood may stimulate mobilization of progenitor cells into circulation. Leukocytapheresis may contribute to tissue repair through promotion of engraftment of progenitor cells to inflamed mucosa. Treatment of CD patients with GM-CSF has been recently reported. GM-CSF is a hematopoietic growth factor that

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stimulates proliferation and activation of granulocytes of the intestinal epithelium (Crosier & Clark, 1992). Impaired innate immune response in granulocytes/macrophages may be an initiating factor in the pathogenesis of CD (Eckmann, 2004), allowing persistent bacterial translocation to the lamina propria and excessive T-cell activation in predisposed individuals, and GM-CSF may therefore improve granulocyte/macrophage function and relieve disease activity (Nagayama & Watanabe, 2005). However, GM-CSF is a potent activator of mutipotential stem cells, including BM-derived stem cells, and drives their mobilisation into the peripheral circulation (Ho et al., 1996; Madero et al., 2000). Thus, GM-CSF may induce mucosal repair through BM stem-cell engraftment and differentiation within mucosal compartments. The combination of G-CSF and GM-CSF has far stronger effects on stem cell mobilization (Madero et al., 2000), and thus a combination of GM-CSF plus G-CSF may optimise treatment of IBD patients. Autologous hematopoietic stem-cell transplantation (HSCT) is an extension of immune modulation/suppression that maximizes immune suppression to the point of immune ablation. In theory, the transplant-conditioning regimen ablates aberrant disease-causing immune cells, whereas hematopoietic stem cells regenerate a new and antigen-naive immune system similar to the normal ontogeny of the immune system during fetal development. Recently, sufficient efficacy of HSCT for CD patients was reported (Lopez-Cubero et al., 1998; Burt et al., 2003; Lashner, 2005), and it therefore appears that BM cell replacement might contribute to induce a naive immune system and improve disease activity. However, our observations that BM transdifferentiation to form nonhaematopoietic lineages is significantly upregulated in response to inflammation (Brittan

Fig. 6. Haematopoietic colony formation in peripheral blood mononuclear cells (PBMC) isolated from active UC patients. Colony assay were performed by methylcellulose method. PBMC were cultured in methylcellulose semiliquid medium in the presence of IL-3, SCF, erythropoietin, G-CSF and GM-CSF for 10 days. In active UC patients, average colony number was 20 ± 5.5/105 cells. (A) Large GM-CSF. M indicates monocytes and G indicates granulocutes, (B) erythroid colony, (C) 2 large GM-CSF and (D) erythroid and GM-CSF.

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et al., 2005) imply that BM cells may also actively contribute to mucosal repair. BM contributes to over 70% of ISEMF in mice with TNBS-induced colitis that have received a lethal dose of irradiation and a BM transplant (Brittan & Wright, 2002). These studies support the potential usefulness of HBST for treatment of IBD, and also suggest a mechanism of action of the transplanted cells in their capacity to transdifferentiate and form nonaematopoietic cells. 10. Conclusion In this review, we summarized recent findings of the role of ISEMF in mucosal inflammatory and repair. There are increasing reports concerning Th-17 cells, a new subclass of helper T cells, in the pathophysiology of intestinal inflammation. IL-17 and Th-17 cells may be a potential target for the treatment of patients with IBD. Furthermore, recent studies suggest that BM-derived cells colonize the intestinal mucosa and differentiate into ISEMF, thereby, controlling mucosal inflammatory and repair responses. This suggests that the ability to regulate stem cell function in normal and diseased tissues would have significant advantages in improving wound healing by accelerating stem cell responses. However, it remains to be seen whether functional differences exist in ISEMF and/or BM stem cells between normal individuals and patients with IBD. Further comparative analysis of these proposals should be performed in the future. References Alison, M. R., Poulsom, R., Jeffery, R., Dhillon, A. P., Quaglia, A., Jacob, J., et al. (2000). Hepatocytes from non-hepatic adult stem cells. Nature 406, 257. Andoh, A., Fujino, S., Bamba, S., Araki, Y., Okuno, T., Bamba, T., et al. (2002a). IL-17 selectively down-regulates TNF-alpha-induced RANTES gene expression in human colonic subepithelial myofibroblasts. J Immunol 169, 1683−1687. Andoh, A., Fujino, S., Okuno, T., Fujiyama, Y., & Bamba, T. (2002b). Intestinal subepithelial myofibroblasts in inflammatory bowel diseases. J Gastroenterol 37(Suppl 14), 33−37. Andoh, A., Hata, K., Araki, Y., Fujiyama, Y., & Bamba, T. (2002c). Interleukin (IL)-4 and IL-17 synergistically stimulate IL-6 secretion in human colonic myofibroblasts. Int J Mol Med 10, 631−634. Andoh, A., Ogawa, A., Kitamura, K., Inatomi, O., Fujino, S., Tsujikawa, T., et al. (2004). Suppression of interleukin-1beta- and tumor necrosis factor-alphainduced inflammatory responses by leukocytapheresis therapy in patients with ulcerative colitis. J Gastroenterol 39, 1150−1157. Andoh, A., Bamba, S., Fujiyama, Y., Brittan, M., & Wright, N. A. (2005a). Colonic subepithelial myofibroblasts in mucosal inflammation and repair: contribution of bone marrow-derived stem cells to the gut regenerative response. J Gastroenterol 40, 1089−1099. Andoh, A., Yasui, H., Inatomi, O., Zhang, Z., Deguchi, Y., Hata, K., et al. (2005b). Interleukin-17 augments tumor necrosis factor-alpha-induced granulocyte and granulocyte/macrophage colony-stimulating factor release from human colonic myofibroblasts. J Gastroenterol 40, 802−810. Andoh, A., Zhang, Z., Inatomi, O., Fujino, S., Deguchi, Y., Araki, A., et al. (2005c). Interleukin (IL)-22, a member of the IL-10 subfamily, induces inflammatory responses in colonic subepithelial myofibroblasts via NFkappaB, AP-1 and MAP-kinase dependent pathways. Gastroenterology 129, 869−884. Bamba, S., Andoh, A., Yasui, H., Araki, Y., Bamba, T., & Fujiyama, Y. (2003a). Matrix metalloproteinase-3 secretion from human colonic subepithelial myofibroblasts: role of interleukin-17. J Gastroenterol 38, 548−554.

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