Tl164

Downregulation of Cytoprotective Intestinal Heat Shock Proteins in Inflamed Mucosa and by InfLammatory Cytokines Maor Lahav, Mark W Musch, ttongyu Ren, leo'old R, Turner, Eugene B. Chang

Background & Aims: Inducd)/e heat shock proteins (hsp) protect viability, and function of intestinal epithelial cells (IEC) against inflammation-associated injury. Unlike small intestine, they are expressed in coloinc IEC under pfiysiological conditions, an eft~:ct dependent on colonic flora as well as mucosal immune cells Because increased hsp expression in inflamed umcosa would be predicted as a host response to limit the extent and severity of mucosal injury, we invrstigated hsp expression in mucosal biopsies from IBD patients, DSS-induced colitis, and in vitro. Methods: Protein expression of inducfble hsps, hsp25/27 and hsp72, and'the constitutively expressed hsc73 were determined by Western blots and immnno- staining (IH) m colonic tnucosa d-ore 1BD patients and DSS-induced colitis. Proqnflannnatory cytokines IL-1~, TNFoc 1FNy were used to elucidate potential factors ahering hsp expression in murine colon-cells (YAMC) and in vivo. Results: Hsp27 and hsp72, but not hsc73, were significantly decreased (66% and 75% respectively) in areas of active inflammation in IBD patients vs uninvolved or healthy' subject samples. When normalized to two epithelial cell markers (cytokeratm 18 and villin), the decreased expression was highly" significant (p<0.01), These changes were paralleled by diminished expression of colonic epithelial cell hsps, assessed by IH. In DSS-induced colitis, a rune-dependent decrease in hsp25 (95%) and hsp72 (53%) protein expression and IH (nadir at day 7) were observed and inversely related to increased myelopenaxidase activuy and histologic grading. By contrast, no changes in mucosal hsc73, villin, or cytokemtin expression were observed. The treatment of YAMC cell lines by I IA~ (5ng/ml) or combined TNFmqFNy (100ng/ml; 200U/relY completely inhibited induced hsp25 and hsp72 expression. In addition, If-l!3 and TNBeFIFNy adminis- tered IP to healthy mice resuked in marked depletion of hsp25 (80%) and hsp72 (91%), but not hse73, expression in colon mucosa Conclusion: Expression of cytoprotective hsps, hsp25 and hap72, are decreased in the areas of active inflammation in colon, an effect mediated in part by pro-inflammatory c3aokines, Such an effect would render the colonic mucosa more susceptible to inflammation-associated injury. Conversely, augmentation of the cytopmtective hsp response by pharmacological, dietary, or probiotic treatment could benefit patients with IBD and other conditions of injured mucosa.

T1165

Endoplasmic Reticulum Stress in Mucosal Smooth Muscle Cells Upregulates Hyaluronan Deposition and Leukocyte Adhesion Alana K, Majors, Richard C, Austin, Carol A. De La Motte, Reed E, Pyeritz, Scott A. Strong

Endoplasmic reticnlum (ERa stress is associated with inflammation, but the relationship between ER stress and inflammatory diseases is not known We have previously shown that the inflammation typical of Crohffs disease and ulcerative colitis is associated with enhanced deposition of hyaluronan (HA) in the extracelkdar matrix of the intestinal mncosa. Although mucosal smooth muscle cells (M-SMCs) are recognized to produce HA, the mechanisms responsible for this abnormal HA accumulauon remain poorly understood. Therefore, we examined the capacity of ER stress to modulate HA deposition by M4MCs in cultures derived from human colon surgical specimens. Visualization of hyaluronan wRh affinity histochemistry and fluorescent contocal microscopy demonstrated little accumulation of HA in untreated cultures. In contrast, M-SMC cultures treated with tunicamycin, an agent that strongly induces ER stress by interkring wi[h glycosylation, demonstrated a matrix rich in HA that was present in both coat and novel, cable-like structures. Thapsigargin and A23187, whmh induce ER stress by altering calcium homeostasis, also upregulated the deposition of HA. Likewise dextran sulfate another agent that induces ER stress, and promotes intestinal inflammation in vivo, dramatically induced HA deposition~ Leukocyte adhesion assays employing radiolabeled U937 cells or peripheral blood mononuclear leukocytes, demon- strated minimal leukocyte binding to untreated M-SMCs, but significantly- increased adhesion (154old) when ER function of the M-SMCs was initially perturbed. The bound leukocytes were released by digestion with hyaluronidase, suggesting HA-mediated adhesion. Fluore> cence micros#opy conhrmed that fhe ttA-containing cables served as attachment sites for the leukocytes This data indicates a novel mechanism exists through which ER stress of M-SMCs induces leukocyte adhesion by enhancing the accumulation of a unique form of hyaluronan and suggests that ER steess may contribute to the pathogenesis of inflammatory bowel disease by ahenng the extracellular mamx and its capacity to interact with leukocytes.

T1166

HIV Infection is Associated with Independently Expanded Populations of Mucosal and Peripheral Blood VS1 Gamma Delta T Cells Michael A, Poles, Shady Barsoum, Patricia Sun, jeanine Daly, David Ho, Linqi Zhang

Background: Gamma Deha (GD) T cells, potentially important components of the innate immune response against HW, are primarily found in the gastrointestinal mucosa. The effect of HW infection on this cellular population in the colonic mucosa has not been previously, studied Methods: We utilized flow cytometry to pbeuotype GD T lymphocytes derived from the peripheral blood and from cokmic biopsies of HlVdntected and uninfected subjects. Relatedness between GD T cell populations in blood and mueosa were determined by analyzing their CDR3 length polymorphisms and by nucleotide sequencing. Statistical com- parisons were made using a paired t-test, Resnha: Chronically HIV-infected subjects, without diagnosed colonic disease, had a significantly greater percentage of T cells expressing the GD T cell receptor in their colonic mncosa compared to healthy subjects (26,] +/ - 184 vs. 132% + / 5 2 % , P= 004) \~&ile in heafihy controls VAI-positive cells accounted for 2 6 6 +/- 21 2% of peripheral blood GD T cells and 41,8 +/- 133% of mucosal GD T cells, they comprised the dominant population of GD T ceils in both the mucosa (58.1 +/- 19 1%) and peripheral blood (602 +/- 110.5%) (For both comparisons, P< 0,001) of HIV- int):cted subjects The expansion of VA1 GD T cells occurred at the expense of the VA2 population which decreased from 422 + / 20,7% to 3.0 +/- 36% of GD T cells in the

blood (P< 0,001) and from 18.8 +/- 6.2% to 10.5 +A 82% of GD T cells in the colonic mucosa (P = 0,01). HIVinfection is associated with an increase in the percentage of peripheral bbod GD T cells that express either the raucosal homing receptors CCR9 or CD103, (37 +/- 2.7% on GD T cells of HW+ subjects vs. 1.2 + / 1.4% on GD T cells of healthy controls (P= 0,01). This is unlikely" to indicate significant Matedness between the GD T cells contained in the blood and colonic mucosal compartments as indicated by significant differences in the patterns of CDR3 length polymorphisms and nucleotide sequences of the VA 1 and VA2 elements of the GD TCR of l~nphoeytes from these sites. Conclusions: Based on these findings we conclude that HIV infection results in expansion of the mucosal GD T cell population, Given the increase in mucosal and peripheral blood VG1 GD T cells, increased expression of mucosal homing receptors may indicate increased homing to or egress from the gastrointestinal mucosa, Still CDR3 length polymorphism and VA element nucleotide sequencing indicate that mucosal and peripheral blood GD T cell populations are distinct.

T l 1 6 7

The Glycoprotein (gp) 96 ls Absent in Intestinal Macrophages from Patients with Inflammatory Bowel Disease (IBD) Katja Schmiter, Martin Hausmann, Werner Falk, Hans Herfarth, Juergen Schoelmerich, Gerhard Rogler

Introduction: Gp96 (Tra-la is an endoplasmatic reticulum-localized chaperone with three known functions: (1) During cellular suzss peptide loaded gp96 can be secreted and internal- ized by antigen presenting cells (APC) via the CD91 receptor. (2) Antigens are then trmrs- ported to the major histocmnpatibdity complex (MHC) class l to be presented to Tseells inducing immunity or tolerance. (3) Secreted gp96 can interact with toll like receptors (TLR) 2 and 4 and activate NF-KB, thus making gp96 the first example for a non pathogen TLRdigand. Methods: CD33 + intestinal macrophages were isolated from intestinal mucosa by immuno- magentic beads. Subtractive hybridization was performed as described. Gp96 mRNA was detected by RT-PCR. Gp96 protein expression was investigated by double labelling immuno- histochemistry in frozen sections from intesnnal mucosa from patients with and without IBD. Results: By a ddte'rential display method we tbund expression of gp96 mRNA in macrophages from not inflamed intestinal mucosa in contrast to in vitro differentiated maerophages, which do not express gp96. In macrophages from not inflamed and from 1BD mucosa gp96 mRNA was detected by PCR confirming these results. Immunohistochemistry further confirmed the expression of gp96 protein in macrophages in not inflamed mucosa. In patients with IBD gp96 protein was absent in intestinal maerophages. Conclusion: The chaperone gp96 is expressed on mRNA and protein level in normal intestinal macrophages, however, gp96 protein is absent in macrophages in IBD mucosa. The lack of gp96 in macrophages from IBD can indicate posttranscriptional regulation, secretion or masking by the bound peptides and be associated with a loss of tolerance against luminal anti- gens.

Supported by the Deutsche Forschungsgemeinschaf~ (SFB 585)

Tl168

The Relevance of the TLR4 Asp299gly Gene Polymorphism in the Susceptibility to Duodenal Ulcer and Severity of Gastric Antral Inflammation Servaas A. Morre, Mathijs P Bergman, Elisabeth Bloemena, Ben J Appehnelk, Christina M Vandenbroucke-Grauls, Amado Salvador Pena

Background & Aims: Recent publications suggest that the Toll-like Receptor 4 (TLR4) contributes to intestinal inflammation and imply that alterations in the innate respm~se systeal may contribute to the pathogenesis of intestinal disorders. We investigated the role of the recently identified functional human TLR4 Asp299Gly gene polymorphism m the susceptibility to duodenal ulcers and the severity of inflammation. Methods: We determined the fi'equency of the TLR4 Asp299Gly gene polymorphism using a restriction fragment length polymorphism based polyTnerase chain reaction (Morre et al J Infect Dis 2002;186:1377) in Dutch Caucasian patients with endoscopicaUy diagnosed duodenal ulcers (DUn = 70) and in heahhy controls (HC n = 175). The frequencies of the wild4ype, heterozygous, and homozygons TLR4 genotypes in the two groups were determined. In addition, histological parameters of inflammation were assessed in antrum biopsy specimens. Results: A two fold increase in the frequency of the TLR4 mutant 2-allele was observed in DU =20.0% versus the healthy controls HC = 10.3% (p = 0.0575 (OR 2.18 (95% CI: 1.02-4.67)). in patients with the TLR4 Asp299Gly mutation the histological scores of inflammation, activity, and atrophy (no inflamntation-mild versus moderate-severe) were higher as compared to patients without this mutation: inflammation 93 vs 74% (p=0.17), activity 57 vs 26% (p=0.062), and atrophy 71 vs 35% (p=0.031). Conclusions: This study, suggests that the functional TLR4 Asp299Gly mutation is involved in DU susceptibility. In addition, patients carrying this mutation bad significant higher scores of histological inflammatory abnormalities in the antrum. Further studies are necessary to clarify' the role that TLR4 plays in the pathogenesis of duodenal and gastric ulcer (GUY. Since the TLR4 tunctions as a LPS receptor and duodenal ulcers are strongly associated with Hdicobacter pylori (HP) we are in progress of detem~inmg both in the GU and DU group the HP status to investigate if the HP status changes our finding on the susceptibility to DU and the severity of gastric inflammation

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