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Necrotic and Apoptotic Intestinal Epithelial Cells Induce Maturation of Dendritic Cells Grzegore W. Telega, Rong Ying

Background and Aims: In our understanding of gut immune system one of the most important questions is how immune system is able to tolerate multiple antigens of commensal gut flora, and on the other hand respond with inflammation to handful of pathogens. Dendritic cells are major antigen presenting cells in the intestinal mucosa. Their activation is prerequisite for antigen presentation and generation of effector T-cells. In our study we tried to find out whether necrosis and/or apoptosis of epithelial cells could activate dendritic cells providing signal for maturation.

Methods: We isolated and cultured primary murine colonic and small bowel epithelial cells using novel technique. Isolated epithelial cells were tested for metabolic integrity and markers for the epithelial cells. Dendritic cells were generated from the bone marrow of the mice by stimulation with GM-CSF. Apoptotic epithelial cells were generated by incubation with IFN-a, followed by TNF-a. Presence of apoptosis was confirmed by DNA content assay and by caspase activity. Repeated freezing and thawing generated necrotic epithelial cells. Den- dritic cells were incubated with necrotic or apoptotic intestinal cells for 12 hour. Intact intestinal cells and culture media served as negative control. After incubation dendritic cells were stained with markers for mature dendritic cells (CD40, CD44, CD80, B7.1, B7.2, MHC-II), than evaluated by flow eytometry.

Results: Necrotic and apoptotic epithelial cells induced expression of maturation markers on the surface of the dendritic cells. Neither intact epithelial cells nor media was able to induce maturation of the dendritic cells. There was no significant difference between colonic epithelial cells and small bowel epithelial cells in their ability to induce maturation of the dendntic cells. Necrotic epithelial cells were more active in inducing maturation of the dendritic cells than apoptotic epithelial cells.

Conclusions: Necrotic and apoptotic intestinal cells are able to induce maturation of den- dritic cells.

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Dendritic Cells (DC) Differ between the Small Intestine and Colon: Expression Of the Chemokine Receptor CCR7 Suggests That Small Intestine DC Traffic Constitutively, Colonic DC Only In Inflammation Angela Jones, Ailsa L Hart, Rachael Rigby, Edward D. A. Westcott, AI Windsor, Stella C Knight, Michael A. Kamm, Andrew J. Stagg

Background: Bacteria and inflammatory cytokines stimulate tissue DC to become immnno- stimulator,/cells. Upregulation of the chemokine receptor CCR7 facilitates mtgration of these mature DC to draining lymphoid tissue. DC from intestinal tissue also migrate in the absence of inflammation and these constitutively trafficking DC may be involved in maintaining tolerance to commensal bacteria. Given the different bacterial milieu in the colon and small intestine, we aimed to determine whether DC in different gut regions differ in their trafficking potential. Methods: Lamina propria mononnclear cells (LPMC) were obtained from biopsy and resection tissue by collagenase digestion. Using flow cytometry, DC were identified in LPMC or whole blood as a CD1 lc + , MHC class II + , Lin-/dim population (Lin: antibodies to CD3, CD14, CD16, CD19 CD34 and CD56). CCR7 was detected with a three layer staining protocol using the monoclonal antibody 2H4. List mode data were analysed using WinList software. Results: CCR7 was not detected on DC from peripheral blood or on DC from any of 12 colonic samples of non-inflamed tissue from control individuals. However, CCR7 was presem on DC from 8 of 8 samples from non-inflamed small bowel (11-82% DC expressing CCR7). These DC, like those from colon, had an immature phenotype, with low levels of CD80, CD86 and CD83. CCR7 expression was induced on colonic DC by maturation of the DC in vitro. Six of 10 freshly isolated samples from the inflamed colonic tissue of ulcerative colitis patients also contained CCR7 + DC (9-40% DC expressing CCR7), possibly indicating maturation of the DC with the capacity to migrate from the inflamed bowel. Conclusions: Immature DC that exp~_,s CCR7, normally present only in small intestine, may be a constitutively trafficking population critical for maintaining immunological non-responsiveness to lumenal contents. By contrast, DC in colon, normally expressing CCR7 only on maturation, may be abnormally activated in ulcerative colitis. Trafficking of these activated DC to lymphoid tissue may contribute to the perpetuation of inflammation.

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Monocytic Leukocytes Preferentially Bind to and are Activated by an Adhesive Hyaluronan Matrix on Polyl:C-treated Mucnsal Smooth Muscle Cells. Matrix Formation and Leukocyte Adhesion are Inhibited by HA Oligomers Carol A. De La Motte, Judith Drazba, Vincent Hascall, Akira Asari, Scott A. Strong

BACKGROUND: Typical changes associated with many inflammatory conditions involve degradation of the stable extracellular tissue matrix and replacement with a provisional, hyaluronan (HA)-containing matrix. Consistently, we observe upregulation of HA in the colonic mucosa involved by active inflammatory bowel disease. This HA matrix can be generated in vitro by mucosal smooth muscle cells (M-SMCs) infected with virus or exposed to viral mimic, and serves as a binding site for CD44 receptors expressed by mononuclear leukocytes. AIMS: We sought to characterize lamina propria mononuclear leukocyte (LPMC) populations that preferentially bind to the induced HA matrix. Additionally, we investigated whether exogenously added small ollgomers (4-14 repeating disaccharide units) of the larger HA gtycosaminoglycan could interfere with leukocyte adhesion, or HA matrix formation in vitro. METHODS: Human M-SMCs and LPMCs were derived from resected colon tissue. LPMC or U937 monocytic cell binding to unstimulated or poly l:C-stimulated M-SMCs was assessed using 51Cr labelled lenkocytes. LPMCs were characterized prior to and after HA adhesion by flow cytometry using the antibodies to CD4, CD8, CD13, CD14, or CD19.

RESULTS: Distinct from other LPMCs, monocytic lineage (CD13+, CD14+) leukocytes tend to preferentially bind to HA (3-4 fold population enrichment). In tum, bound monocytes demonstrate CD44 capping and HA internalization. Oligomers (4-14reefs) of HA interfere in a size-dependent manner with both formation of the adhesive HA matrix (35-85% inhibition of adhesion between 4mer and 14mer) and interaction of leukocytes (~55%) with an already formed adhesive matrix. CONCLUSIONS: Hyaluronan-mediated mononuclear leukocyte adhesion represents a non-cytokine-induced pathway of leukocyte recruitmen9 retention by M-SMCs. This mechanism favors interaction with newly recruited monocytes and induces changes in the leukocyte suggestive of cell activation. Small oligomers of the constituent hyaluronan disaccarides effectively interfere with HA matrix formation, and their therapeutic value might warrant future consideration.

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Identification of VCAM-1 + Mesenchymal Cells as a Third Cell Type in Intestinal Cryptopatches Rebekah T. Taylor, Andreas Luegering, lfor Williams

Purpose: Cryptopatches (CP) are aggregates of lymphocyte precursors that develop posmatally in mouse small and large intestine. CP have been proposed as the site of extrathymic differentiation of specific subsets of imraepithehal lymphocytes, but the celfuhr interactions responsible for the genesis of CP have not been defined. Two types of cells have been identified within cryptopatches: Thy-1 + , c-kit + T cell precursors located centrally and CD11c + dendritic cells concentrated around the periphery. The purpose of this study was to determine if VCAM-1 + mesenchymal cells phenotypically similar to those found in the earliest recognizable Feyer's patches were present in CP. Methods: Horizontal frozen sections

,of small intestinal tissue containing CP were stained with antibodies to VCAM-1, CDllc, CD31, MAdCAM-1 and Thy-1. Tyramide signal amplification was used to increase the intensity of VCAM~I and MAdCAM-1 staining. Some sections were double stained for VCAM-1 together with CDl lc or CD31. Fluorescence images were acquired on a Zeiss LSM510 confocal microscope Results: All intestinal CPs examined included multiple VCAM- 1 +ce l l s at the periphery, although the VCAM-1 + cells were typically restricted to only part of the circumference of CP rather than encircling the entire CP. Double staining experiments demonstrated that the VCAM-1 + cells were a distinct set of cells from the CD1 lc + cells principally found all around the central area of T cell precursors. VCAM- 1 + cells around CP were not CD31 + , while all VCAM-1 + vascular structures in the lamina propria also co-expressed CD31. No vascular structures expressing CD31, MAdCAM, or VCAM-1 were identified within CP. Conclusion: We have identified VCAM-1 + cells as a third major cell type present in intestinal cryptopatches in mice. The VCAM-1 + cells are mesenchymal cells that do not express any endothelial cell markers including CD31. We propose that the VCAM-1 + ceils in CP may play a similar organzing role in the genesis of CP as do the VCAM-1 + cells that cluster in the iutestinal wall in late prenatal life as the first step in the development of Peyer's patches.

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CD28 regulates proximal T cell signaling events Pietro G. Andres, Kimberly C. Howland, Abul K. Abbas, Mathew Krummel

Most animal models suggest that dysregulated activation and differentiation of CD4 + T cells are critical to the pathogenesis of inflammatory bowel disease. The costimulatory molecule CD28 is a key regulator of CD4 + T cells. However, when and how this molecule affects the T cell-antigen presenting cell (APC) interaction is unclear. We used real-time molecular imaging to determine the role of CD28 in proximal T cell signaling events. METHODS: To visualize CD28 at the immunological synapse we transfected a GFP-tagged CD28 construct into the D10 T cell line, which was then imaged interacting with the B cell lymphoma line CH27 loaded with cognate antigen. To determine the role of CD28 in driving T cells to flux calcium, we loaded CD4+ T cells from wild-type and CD28-/- DOl1.10 mice with the calcium dye Fura-2, and imaged them while they were interacting with I-Ad + B7-transfected, OVA-pulsed CHO ceils. To determine the effects of costimulation in cis versus trar~s, CD28-deficient T cells were reconstituted with CD28 Y170F mutants and restimulated with CHO cells transfected with l-Ad with or without CHO cells transfected with B7 or with CHO cells transfected with both I-Ad and B7. T cell proliferation and IL- 2 production were used as read-outs. RESULTS: Prior to contact with an APC, CD28 is distributed evenly across the surface of the T cell. Afterwards, CD28 rapidly moves to the point of contact between T cell and APC. This movement precedes calcium flux. Compared to wild-type cells, calcium flux is 3-fold reduced in CD28-/- cells, reflecting a defect in recruiting extracelfular calcium stores. Given the rapid clustering of CD28 in the T cell- APC contact zone, we reasoned that one function of CD28 is to recruit signaling molecules to the synapse. To test this hypothesis, we examined the ability of CD28 cytoplasmic domain mutants to function independently of the T cell receptor. We have previously shown that a Y170F mutant of CD28, which cannot bind PI3K, still drives T cell activation. We thus asked if the Y170F construct could signal when ligated in trans or in cis with the TCR. Compared to a wild-type CD28 construct, the Y170F mutant was unable to restore IL-2 production when stimulated in trans, but functioned normally in cis. CONCLUSIONS: CD28 migrates rapidly to the immunological synapse and functions early in the T cell-APC imeraction. While CD28 may initiate some signals independently of the T cell receptor, its principal role may be to provide a site, its cytoplasmic tail, for co-localization of signaling mol- ecules.

AGA Abstracts A-154