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Background & Aims: Inflammatory bowel disease (IBD) is associated with increased apoptosis of intestinal epithelial cells (IECs). Mutations in the tumor suppressor p53 appear during early stages of progression from colitis to cancer. We investigated the role of p53 and its target, p53-upregulated modulator of apoptosis (PUMA), in inflammation-induced apoptosis of IECs., Methods: Apoptosis was induced in mouse models of mucosal inflammation. Responses of IECs to acute, T-cell activation were assessed in wild-type, p53⁻/⁻, Bid⁻/⁻, Bim⁻/⁻, Bax3⁻/⁻, Bak⁻/⁻, PUMA⁻/⁻, and Noxa⁻/⁻ mice. Responses of IECs to acute and chronic colitis were measured in mice following 1 or 3 cycles of dextran sulfate sodium (DSS), respectively. Apoptosis was assessed by TUNEL staining and measuring activity of caspases 3 and 9; levels of p53 and PUMA were assessed in colon tissue from patients with and without ulcerative colitis., Results: Apoptosis of IECs occurred in the lower crypts of colitic tissue from humans and mice. Colitis induction with anti-CD3 or 3 cycles of DSS increased apoptosis and protein levels of p53 and PUMA in colonic crypt IECs. In p53⁻/⁻ and PUMA⁻/⁻ mice, apoptosis of IECs was significantly reduced but inflammation was not. Levels of p53 and PUMA were increased in inflamed mucosal tissues of mice with colitis and in patients with UC, compared with controls. Induction of PUMA in IECs of p53⁻/⁻ mice indicated that PUMA-mediated apoptosis was independent of p53., Conclusions: In mice and humans, colon inflammation induces apoptosis of IECs via p53-dependent and - independent mechanisms; PUMA also activates an intrinsic apoptosis pathway associated with colitis., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Mechanisms responsible for crypt architectural distortion in chronic ulcerative colitis (CUC) are not well understood. Data indicate that serine/threonine protein kinase Akt (Akt) signaling cooperates with Wingless (Wnt) to activate beta-catenin in intestinal stem and progenitor cells through phosphorylation at Ser552 (P-beta-catenin(552)). We investigated whether phosphoinositide 3-kinase (PI3K) is required for Akt-mediated activation of beta-catenin during intestinal inflammation., Methods: The class IA subunit of PI3K was conditionally deleted from intestinal epithelial cells in mice named I-pik3r1KO. Acute inflammation was induced in mice and intestines were analyzed by biochemical and histologic methods. The effects of chemically blocking PI3K in colitic interleukin-10(-/-) mice were examined. Biopsy samples from patients were examined., Results: Compared with wild-type, I-pik3r1KO mice had reduced T-cell-mediated Akt and beta-catenin signaling in intestinal stem and progenitor cells and limited crypt epithelial proliferation. Biochemical analyses indicated that PI3K-Akt signaling increased nuclear total beta-catenin and P-beta-catenin(552) levels and reduced N-terminal beta-catenin phosphorylation, which is associated with degradation. PI3K inhibition in interleukin-10(-/-) mice impaired colitis-induced epithelial Akt and beta-catenin activation, reduced progenitor cell expansion, and prevented dysplasia. Human samples had increased numbers of progenitor cells with P-beta-catenin(552) throughout expanded crypts and increased messenger RNA expression of beta-catenin target genes in CUC, colitis-associated cancer, tubular adenomas, and sporadic colorectal cancer, compared with control samples., Conclusions: PI3K-Akt signaling cooperates with Wnt to increase beta-catenin signaling during inflammation. PI3K-induced and Akt-mediated beta-catenin signaling are required for progenitor cell activation during the progression from CUC to CAC; these factors might be used as biomarkers of dysplastic transformation in the colon., (Copyright © 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Mesalamine is a mainstay therapeutic agent in chronic ulcerative colitis (CUC) in which condition it reverses crypt architectural changes and reduces colitis-associated cancer (CAC). The present study addressed the possibility that mesalamine reduces beta-catenin-associated progenitor cell activation, Akt-phosphorylated beta-catenin(Ser552) (P-beta-catenin), and colitis-induced dysplasia (CID)., Methods: Effects of mesalamine on P-beta-catenin staining and function were assessed by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) in biopsy specimens of CUC in mild or "refractory" severe mucosal inflammation. Effects of mesalamine on epithelial proliferation and activation of Akt and beta-catenin were assessed in interleukin (IL)-10(-/-) colitis and CID by immunohistochemistry and Western blotting. Dysplasia was assessed by counting the number and lengths of lesions per colon., Results: Data from IL-10(-/-) and human colitis samples show that mesalamine reduced Akt activation and P-beta-catenin levels in the middle and upper crypt. Reductions in P-beta-catenin in CUC biopsy specimens with severe inflammation suggested that mesalamine reduced P-beta-catenin levels in tissue refractory to mesalamine's anti-inflammatory effects. In IL-10(-/-) mice, mesalamine reduced CID concordant with inhibition of crypt Akt and beta-catenin signaling., Conclusions: The results are consistent with the model that mesalamine contributes to chemoprevention in CAC by reducing beta-catenin signaling within intestinal progenitors.

Background & Aims: Chemokines mediate cell trafficking in inflammatory states such as allograft rejection. However, their role in small-bowel allograft rejection has not been defined. The aim of this study was to examine the roles of type 1 helper T-cell chemokines in small-bowel allograft rejection., Methods: Mucosal histology, chemokine messenger RNA (real-time polymerase chain reaction), and cell isolates were examined in small-bowel allografts and isografts. Interferon-gamma-inducible protein-10/CXC chemokine receptor (CXCR) 3 interactions were specifically evaluated by using allografts from interferon-gamma-inducible protein-10(-/-) donors and adoptive transfer of CXCR3(-/-) T cells into recombination activating gene (RAG)-1(-/-) recipients of small-bowel allografts., Results: Type 1 helper T-cell cytokine (interferon-gamma) and chemokine (interferon-gamma-inducible protein-10, monokine induced by interferon-gamma, macrophage-inflammatory protein-1 alpha, and regulated on activation, normal T cells expressed and secreted) messenger RNA up-regulation was detected (real-time polymerase chain reaction) by postoperative day 3 in small-bowel allografts. Interferon-gamma-inducible protein-10(+/+) small-bowel allograft rejection was associated with a dramatic (>7-fold) increase in CXCR3(+) host T cells in the graft lamina propria. With interferon-gamma-inducible protein-10(-/-) small-bowel allografts, CXCR3(+) host T-cell infiltration of the graft lamina propria was markedly decreased and rejection was significantly delayed. Whereas adoptive transfer of wild-type B6 (CXCR3(+/+)) T cells into B6 (RAG-1(-/-)) recipients induced rapid rejection of CB6F1 small-bowel allografts, rejection was significantly delayed (29.2 +/- 8.7 days vs. 16.5 +/- 3.1 days; P < 0.01) in B6 (RAG-1(-/-)) mice reconstituted with T cells from B6 (CXCR3(-/-)) mice., Conclusions: Recruitment of CXCR3(+) host T cells by donor derived interferon-gamma-inducible protein-10 may precipitate small-bowel allograft rejection. These data highlight the importance of type 1 helper T cell-related chemokines in promoting cell-mediated rejection responses in small-bowel allografts and suggest that interferon-gamma-inducible protein-10 is an attractive therapeutic target for humanized monoclonal antibody strategies.

Background & Aims: Previous data suggest that oral antigen induces interferon (IFN)-gamma production in intestinal T cells. However, oral tolerance is associated with decreased production of IFN-gamma by T cells after antigen sensitization. The aim of this study was to examine the role of IFN-gamma in oral tolerance., Methods: Oral tolerance was examined in BALB/c mice after the adoptive transfer of T cells from chicken ovalbumin (OVA(323-339))-specific, DO11.10 x RAG-1(-/-) T-cell receptor transgenic mice., Results: OVA feeding induced systemic tolerance of delayed-type hypersensitivity (DTH) and antibody responses. OVA feeding up-regulated IFN-gamma production by transgenic T cells in Peyer's patch and mesenteric lymph node but not splenic tissues. Treatment of OVA-fed mice with neutralizing monoclonal antibody to IFN-gamma prevented tolerance of DTH responses. Analysis of transgenic T-cell numbers in DTH sites by immunohistochemical staining suggested that induction of IFN-gamma by oral antigen decreased accumulation of transgenic T cells in cutaneous sites of antigen injection. IFN-gamma-deficient or wild-type DO11.10 and BALB/c mice were used to show that IFN-gamma production by donor transgenic T cells was critical for oral tolerance., Conclusions: These data suggest that the induction of IFN-gamma by oral antigen contributes to systemic tolerance by decreasing migration of T cells to peripheral sites of inflammation.

Background & Aims: The intestinal epithelial compartment is populated by CD8(+) alpha beta and gamma delta intraepithelial lymphocytes (IELs), which monitor the integrity of the epithelial barrier. alpha beta IELs are activated by peptide antigens presented by class I major histocompatibility complex (MHC) molecules, but it is unclear how gamma delta IELs are activated., Methods: G8 T-cell receptor (TCR) gamma delta transgenic (Tg) mice (specific for the class I MHC alloantigen, T22/10(b)) were crossed to class I MHC-deficient beta2-microglobulin-knockout (beta2m degrees) mice, and Tg+ IELs were examined for relative yields and surface and functional phenotype., Results: Evidence for class I MHC-induced activation of Tg+ IELs was supported by the detection of 4-fold greater numbers of Tg+ IELs in G8 x beta2m+ mice that proliferated at 15-fold higher levels than IELs from G8 x beta2m degrees mice. However, expression of CD69, production of cytokine (interleukin 2 and interferon gamma), and detection of cytolytic function for IELs in G8 x beta2m degrees mice suggested that class I MHC was not required for gamma delta IEL development or maturation., Conclusions: These results suggest that CD8(+) TCR gamma delta IELs do not require class I MHC for development but support the notion that antigens presented by class I MHC molecules are involved in the peripheral expansion and differentiation of this subset.