Genes expressed in non-transformed human intestinal primary epithelial cell (HIPEC) lines derived from patients with IBD and controls

the following concentrations: DCA and CDCAat 250-300/~M. At higher concentrations toxic effects were observed. The maximal IL-8 induction in responseto TDCA was measured at a concentration of 2mM, without cytotoxic effects. IL-8 RNAquantification confirmed the protein data. Conclusion: Chemokine expression in a human colonic epithelial cell line is increased in responseto different bile acids. This may be a novel mechanism initiating and aggravating inflammation in the colon and might be associatedwith the promotion of neoplastictransformations in colitis via perpetuating the inflammation and promoting cell proliferation.

3750 Oxidative Stress Sensitizes Murioe Intestinal Epithelial Cells To Immune-Mediated Damage: Role Of Fas Death Receptor In Apoptnsis Timothy L. Denning, Hiromasa Takaishi, Sheila E. Crowe, Istvan Boldogh, Univ of Texas Medical Branch, Galveston,TX; Anthony Jevnikar, Univ of Western Ontario, London Canada; Peter B. Ernst, Univ of Texas Medical Branch, Galveston, TX Background: Examinationof colonic tissue from humans with IBD or in animal models of colitis shows that activated neutrophils are adjacentto intestinal epithelial cells during active disease. Neutrophils impart an oxidative stress to adjacent cells that alters the expression of genes associatedwith the induction of apoptosis. In this study, gene expressionand apoptosis were measured in intestinal epithelial cells following exposure to oxidative stress. Methods: Intestinal epithelial cell lines (Mode-K and IEC-4.1) were cultured in the presence of varying concentrations of a representativeoxidative metabolite, H202.Apoptosis was monitored using the ELISAand JAM assaysto detect DNA fragmentation. Real-timeand conventional RT-PCR, RNA protection assays, flow cytometry and western blots were used to assaythe expression of Fas death receptor and Fas-L. The role of Caspase-8 in cell death was addressed using the specific inhibitor, z-lETD-fmk. Results: Cells cultured in the presence of H202showed an increase in the expression of mRNA and protein for Fas and FasL. The change in Fas and FasL expressionwas associatedwith an increase in apoptotic index from 1 to a mean of 7.5 in Mode-K and 6.4 in IEC-4.1 after exposureto 200/LM H202.TOassessthe affect of oxidative stress on lymphoepithelial cell interactions, epithelial cells exposed to 150/~M H202were stimulated with an agonistic anti-Fas antibody that would mimic FasL-mediated killing by adjacent T cells. The apoptotic index was 4.5 or 4.1 after exposureto the isotype control and increased to a mean of 16.3 or 37.2 in Mode-K and IEC-4.1 cells respectively in responseto anti-Fas. Furthermore, activated splenocytesand colonic lymphocytes induced Fas-mediated DNA fragmentation of intestinal epithelial cells. The increase in Fas expression and the sensitivity to apoptosis was inhibited up to 80% when antioxidants were administered with the HzOz.Inhibition of caspase-8,an essentialcaspasein the Fas-mediatedapoptotic cascade, significantly blocked H202-inducedapoptosis, suggesting a direct role for Fas in oxidative stress-mediatedapoptosis. Conclusions:Oxidativemetabolitesdirectly induce Fas, Fas-L, and apoptosis in intestinal epithelial cells. Oxidative stress further sensitizes the epithelium to immune-mediated damage. These data support the hypothesis that oxidative stress may "prime" enterocytesfor direct and indirect modes of apoptosis thus exacerbatingintestinal inflammation.

3751 Keratinocyte Growth Factor Binds To Collagens Of The Gastrointestinal Tract: Identification Of The Collagen Binding Structure Martin Ruehl, Ines Schoenfelder, Clin Benjamin Franklin, Berlin Germany; Richard Farndale, Graham Knight, Dept of Biochemistry, Cambridge United Kingdom; Rajan Somasundaram, Monika Schmid, RenateAckermann, Ernst O. Riecken, Clin Benjamin Franklin, Berlin Germany; Detlef Schuppan, Med Clin I, Erlangen Germany Background/Objectives:Keratinocyte growth factor (KGF) belongs to the family of heparinbinding fibroblast growth factors with a paracrine proliferative effect on intestinal/hepatic epithelial cells. KGF appearsto have an important role in intestinal/liver regeneration. Since after partial hepatectomythe amount of KGF-mRNAdoes not increasesignificantly, an extracellular pool of KGF has been postulated.We could show that KGF binds to collagens. Synthetic tripelhelical collagen peptides with the structure (GPP)x or (GPO)x (0 = Hyp) were used to define potential collagen binding sites. Methods: Collagenous,triple helical synthetic peptides with the structure (GPP)~o,(GPO)~oand the linear control peptide (GAP)~owere immobilized on polystyrene microtiter plates or nitrocellulosefor binding experimentsand dot blots, resp. For inhibition studies microtiter plates were coated with various native collagens (200 ng/ well) and collagen peptides (30-60 ng/well) and incubatedwith 2 ng [~251]-KGFtogether with different concentrationsof the synthetic peptidesfollowed by washing and scintillation counting. Results: 20-25% of radiolabelled KGF bound to the triple helical synthetic peptides, comparable to the binding to collagen CVl or the C-terminal peptide al(I)CB6 of collagen I. Binding studies and dot blot analysis showed a clear preference of KGF binding to (GPO)~o over (GPP)~0demonstrating the importance of hydroxyproline for KGF binding. KGF binding to c~ICB6was inhibited by >50% with a GO-fold molar excess of (GPO)10,whereasthe linear control peptide was not inhibitory. KGF binding to (GPO)~owas blocked by a 3-fold excess of the peptide.Conclusions:KGFbinds specificallyto liver collagensvia collagenousconsensus sequences. The smallest binding structure is a decamertriplet with the structure (GPO)~o.In case of intestinal/hepatic damage, collagen-bound KGF can be rapidly liberated from the extracellular (collagen) steres. The application of peptidic KGF-binding antagonists in vivo could be used to modulate intestinal/liver regeneration.

3752 Epithelial Gene Induction of the Scavenger Receptor Cycteine-Rich (SRCR) Superfamily in Experimental and Human Inflammatory Bowel Disease (IBD) Hitosbi Yonezawa,Kohei Fukushima, Hitoshi Ogawa, Taku Kitayama,Hiroo Naito, Yuji Funayama, Chikashi Shibata, Akihiko Hashimoto, Tohoku Llniv Graduate Sch of Medicine, Sendal Japan; Masanori Terashima, KazuyoshiSaito, Iwate Medical Univ, Morioka Japan; Seiki Matsuno, Iwao Sasaki, Tohoku Univ Graduate Sch of Medicine, Sendal Japan Background and Aims: Bacterial reconstitution (8R) model enables us to detect genes which maintain mucosaldefenseand havean active role in bacteria-associatedmucosal inflammation (GE:118:A720,A786,AJP:279:G492). BecauseGRP-ductin (CRP-D) mRNA was identified after bacterial challenge to germ-free (GF) mice, CRP-ductin and its human homologue, deleted in malignant brain tumors 1 (DMBT1), may be involved in mucosal inflammation. The aims of the present study were to investigate epithelial expression of CRP-D and DMBT1 in experimental and human IBD and their possible role in IBD pathology. Methods: GF mice were orally given bacterial suspension preparedfrom specific pathogen-free mice. Epithelial cells (EC) were isolated and RNA extracted. Differential gone expression was investigated by cDNA microarray and confirmed by northern blotting. Dextran sulfate sodium (DSS)-colitis was developedby free accessof water containing 3% DSSfor 9 days to evaluateECexpression of CRP-D mRNA. DMBT1 expression was also investigated using isolated EC RNAsfrom 18 control, 19 Crohn's disease (CD) and 25 ulcerative colitis (UC) specimens including paired samplesfrom inflamed (Inf) and non-inflamed (N-Inf) areaby northern blotting and quantitative real-time RT-PCR. To analyze DMBT1 induction mechanisms, HT29 and T84 cells were stimulated by IL-lb, -6 and LPS, RNA extracted,then DMBT1 mRNAwas quantitated.Results: Induction of CRP-D mRNA was detected and confirmed in BR as described. This gene was also induced in DSS-colitis even after recovery from active inflammation by the removal of DSS. Northern blotting revealedthat DMBT1 mRNAwith 7 kb was weakly expressedin control and remarkably induced in two-third of IBD EC. An additional 6.5 kb band was noticed in one-third of IBD EC. Real-timeRT-PCRdemonstratedthat DMBT1 mRNAlevelwas significantly (p=O.O02) higher in both UC and CD. When samples from Inf and N-Inf muosae were compared, epithelial expression of DMBT1 from Inf mucosae was unexpectedlyless than NInf mucosae. IL-lb, -6 and LPS failed to induce DMBT1 mRNAin vitro. Conclusion:We clearly demonstrated that CRP-D and DMBT1, the members of SRCR superfamily, are involved in experimental and human IBD. Induction of DMBT1 was not UC- or CD-specific and did not necessarily correlate to the degree of active inflammation, suggesting that DMBT1 may play a role in recoveringprocess and/or down-regulation of immune responsein gut inflammation.

3753 Component Expression AlteraUons Of The Supramucnsal Defence Barrier In Ulcerative Colitis R. J. Longman, Bristol Royal Infirmary, Bristol United Kingdom; R. Poulsom, Histopathology Unit, Imperial Cancer Research Fund, London United Kingdom; B. F. Warren, Dept of Cellular Pathology, John Radcliffe Hosp, Oxford United Kingdom; N. A. Wright, Histopathology Unit, Imperial Cancer Research Fund, London United Kingdom; A. P. Corfield, Univ Dept of Medicine, Bristol Royal Infirmary, Bristol United Kingdom; i . G. Thomas, Univ Dept of Surg, Bristol Royal Infirmary, Bristol United Kingdom Background and Objectives Mucin glycoproteins and trefoil factor family (TFF) peptides play a pivotal role in gastrointestinal mucosal dctence and repair. The aim of this study was to identify alterations in gene expressionand product Iocalisation patterns of these components of the supramucosaldefencebarrier in ulcerativecolitis (UC). Methods Rectosignoidmucosal biospies were obtained in a prospectivefashion from patients with clinically mild UC (n = 13), moderate UC (n=11} and severe UC (n=16) according to the Truelove and Wilts criteria of disease severity. Biopsies were also obtained from patients without identifiable colorectal diseaseas controls (n = 17). Histologicalseverity of UCwas determinedon all diseasebiopsies according to the Bristol index. In situ hybridisation and immunohistochemsitrywere employed to detect TFF peptide (TFF1-3) and mucin (MUC1-6) geneexpressionand product Iocalisation. Semi-quantitativescoring was on an analoguescale (0 to 3 for immunohistochemicalstaining intensity and mRNA detection; 0 to 4 for proportion of epithelial cell populations exhibiting immunohistchemical staining) by two independent observers. Results Clinical severity of disease was strongly correlated with that of histological severity (Kendall's tau-a=O.4218, tau-b=0.5733; p
3754 Genes Expressed In Non-Transformed Human Intestinal Primary Epithelial Cell (HIPEC) Lines Derived From Patients With IBD And Controls Ansamma Joseph, Asit Panja,Winthrop Univ Hosp, Mineola, NY BACKGROUND:Analytical gene expression studies have identified specific genes expressed in several pathological disorders. In both ulcerative colitis (UC) and Crohn's disease (CD), aberrant epithelial cell function may play an important role in mediating inflammatory responses. However,no comprehensivestudy has beenconductedto identity specific changes in gene expression in inflamed vs. uninflamed epithelial cells in IBD probably due to the lack of non-transformed primary cells. We have recently established a number of cultures of pure populations of non-transformed HIPEC lines in long-term culture (Panja, Lab Invest 2000, 80:1473). AIM: To establish an epithelial cell gene expressionprofile in UC and CD, we have analyzed gene expression in a panel of HIPEC lines derived from actively inflamed and uninflamed tissues. METHOD:Total RNA was extracted from confluent monolayersof HIPEC

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lines (normal, UC and CD, grown under identical conditions) and hybridized to microarray ExpressChipTME02 (Mergen Ltd, San Leandro, CA) consisting of 1154 cDNA representing the families of cytokine/receptor, surface antigens, matrix metalloproteinase, proteases, and oncogenes. After normalization, differences in gene expression was analyzed by comparing results from IBB HIPECs with results obtained from a normal control HIPEC line using ExpressData software. Expression of selected proinflammatory genes was further validated by measuring protein synthesis by ELISA. RESULTS: Compared to normal controls, gone expression increased or decreased in 30 genes in UC and in 38 in CD. The major findings (with changes > 5 fold as shown in parenthesis) are as follows: a)360 Increased in UC: MIP3e (41.5), RANTES (64.9), Miple (14.2) HLA-F (6.8), b) Increased in CD: TIM (164), SFRP1 (9.1), Rho-GTPaseactivation Protein (7.1), Rho GDI (18.9). c) Decreasedin UC: CD51 (30.2), HLA-B 12.6, HLA-DP-B1 (5.5), SOD3 (283.4), clusterin (5.3), rl) Decreased in CD: RPL32 (13.3), MYBL-1 (9.5), HLA-DP-B (40.7) and granulocyte chemofachc protein - 2 (5.2). In addition, several genes (MMP1, MIP3, GSI-F1, APOC1, ILRL1) were increased (>6 fold) in both UC and CD. No overlap of underexpressed genes was seen between these two inflammatory conditions. CONCLUSION:Alteration in epithelial cell gone expression occurs in both UC and CD. Further studies with additional HIPEC lines derived from inflamed and uninflamed tissue may help in the development of targeted therapies for IBD.

Intestinal Chloride Secretion Is Down-Regulated In Response To Metabolic Stress Karen L. Madsen, Anthony Cornish, Paul R. Soper, Marianna Kulka, Dean Bolus, Christine Yachimec, Thomas Churchill, Univ of Alberta, Edmonton Canada AMP-kinase has been shown to directly inhibit epithelial CFTR conductance in response to cellular metabolic stress. We have previously shown that inflamed colons from IL-IO KO mice exhibit altered metabolism which can be rapidly corrected by the inhibition of poly-(ADPribose) polyrnerase (PARP). The aim of this study was to determine the role of AMP-kinase and alterations in cellular metabolism in regulating chloride secretion and CFTR expression. Method~. In vivo: IL-tO KO mice were injected ip with the PARP inhibitor, 3-aminobenzamide (3-AB, 20 mg/kg) and control mice with AICAR (250 mg/kg), a specific inducer of AMPkinase(n=6). After 30 min, colons were removed and short circuit current (Isc), Alsc to 104 M forskolin (as a measurement of chloride secretion)and chloride flux were assessed in Ussing chambers. /n vitro: T~ monolayers were exposed to 5 ng/ml IFN-y, or 1 mM AICAR for 48 hrs (n=8). Monolayers were assessed for cellular ATP, CFTR expression by flow cytometry, and ion transport. Resutt~ In viva. IL-IO KO mice demonstrated a 70% reduction in colonic Isc and z~ Isc response to forskolin compared with control mice. Inhibiting PARP activity with 3-AB altered cellular metabolic activity and restored Isc responses within 30 rain (p
3755 Altered Muc2 Synthesis and Sulfntion in Bacterial Induced Colitis in IL-lO -~- Mice Nicoie M. J. Schwerbrock, Fengling Li, Ctr for Gt Biology and Disease, Univ of North Carolina, Chapel Hill, NC; Hans A. Bueller, Sophia Children's Hosp, Rotterdam Netherlands; Alaxandra W. C. Einerhand, Lab Pediatric, Erasmus Univ, Rotterdam Netherlands; R. Balfour Sartor, Ctr for GI Biology and Disease, Univ of North Carolina, Chapel Hill, NC; Jan Dekker, Lab Pediatric, Erasmus Univ, Rotterdam Netherlands Background/Aims: Germ-free IL-IO deficient mice develop colitis only after introduction of enteric bacteria (1]. Mucins protect the vulnerable colonic epithelium and coetTiboteto barrier function. In active ulcerative colitis we demonstrated a decrease in production and sufiaffon of the major mucin MUC2 [2,3]. We now sought to elucidate the kinetics of MUC2 synthesis and sulfation during onset and progression of colitis in IL-lO -~- mice following introduction of enteric bacteria. Methods: Germ-free 129 IL-IO -~- mice were colonized with pathogen free murine enteric bacteria for 1-6 weeks. Colitis was quantified by blinded histolooic score and IL-12 secretion at O, 1,2 and 6 weeks, and Muc2 synthesis was assayed qua.iiiaibc~y (mRNA, biosynthesis, secretion) and histologically (immunehistochemistry, alcian blue/PA.~ining) in the cecum and colon. Results: Germ-free mice were free of colitis, whereas the introduction of SPF bacteria led to very high colonic IL-12 secretion and decreased body weight with progressively worsening of inflammation beginning at 1 week. Mucosal morphology changed dramatically, yet there were still numerous goblet cells staining positive for Mec2 protein. Muc2 mRNA levels showed little change. Yet, Muc2 precursor synthesis increased a least lO-fold within 1 week, then the Muc2 precursor synthesis decreased to control levels. Total Muc2 decreasedgradually over 2 weeks, particulady in the cecum and then returned to control values. Cecal Muc2 secretion decreased by 1 week, then increased more than 2-fold by 2 weeks, but stabilized at control levels by 6 weeks. Sulfation of Muc2 progressively decreased, particularly the Muc2 secreted by the distal colon was very poorly sulfated. Each of the effects on Muc2 expression was specific for IL-IO ~- mice, as these effects did not occur in wild type germ-free mice upon introduction of the same enteric bacteria. Conclusions: Bacteria rapidly induced inflammation in IL-IO ~ mice associated with an early surge in Muc2 precursor synthesis, yet as colitis progressed the Muc2 synthesis normalized, whereas Muc2 sulfation decreasedprogressively. The latter phenomenon was also observed in human ulcerative colitis suggesting that aberrant Muc2 synthesis and sulfation is either primary or secondarily involved in the pathogenesis of colonic inflammation. 1. Sellon et at., Infect Immun 1998, 66:5224-31 ; 2. Tytgat et at., Biochem Biophys Res Comm 1996, 224: 397-405; 3. Van Klinken et al., Gut 1999, 44: 387-93.

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ComMffuthmlyAcffve PhnsphaUdlyinositol 3-Klnase (PI 3-K) and Aid are Sufficient to Stimulate NHE3: Insights In Mechanisms of NHE3 Stimulation Whsseon Lee Kwon, Johns Hopkins Univ Sch of Medicine, Baltimore, MD; David Johns, Megan Caret, Joseph Park, JHUSOM, Baltimore; Philip Tsichlis, Kimmel Inst; T Jefferson Un, Philadelphia, PA; Mark Donowitz, JHUSOM, Baltimore The epithelial brush border Na/H exchanger3(NHE3) is involved in regulated Na absorption in intestine and kidney. The mechanism of NHE3 regulation involves trafficking on and off the plasma membrane. Under both basal and growth factor stimulated conditions, PI 3-K is involved in NHE3 regulation with the PI 3-K inhibitor wortmannin decreasing NHE3transport activity and the amount of NHE3 on the plasma membrane. While PI 3-K was shown to be necessary for NHE3 regulation, the studies relied on inhibitors and the signals downstream of PI 3-K were not identified. The current studies investigated the role of PI 3-K and Akt, a ser/thr kinase which acts downstream of PI 3-K, in regulation of NHE3. Studies used transient expression of constitutively active PI 3-K and Akt. Somatic gene transfer was performed using replication deficient adenovirus containing either a constitutively active form of the catalytic subunit of PI 3-K (p110*-myc) or constitutively active myristilated Akt (myr-Akt-HA). An ecdysone-inducible promoter was used to allow time limited control of gone expression based on addition of the ecdysone receptor agonist ponasterone A. Infection occurred in 100% of PS120/NHE3 cells 48h after infection and 24h after ponastemne. Infection with p110*-myc and myr-Akt-HA followed by ponasterone A increased the amount of PI 3-K and Akt -4 fold each and increased the PI 3-K activity to 223% + / - 16% of control and the Akt activity to 310% + / - 31 of control. Both constitutively active PI 3-K and Akt significant stimulated NHE3 activity, increasing the NHE3 Vmax to 122% + / - 4% (p
3756 Rapid Induction Of The Antioxidant, Heme Oxygeoase-1 (HO-1) In Human iit~Bnal Epithelial Ceils: Interaction With Glutathione Levels Csaha Varga, Maryan Cavicchi, Dominique Lamarque, Jean Charles Delchier, Inserm U99, Creteil France; Brendan J. Whittle, William Harvey Research Institute, London United Kingdom HO-1 is an inducible antioxidant enzyme which can be expressed after exposure to heavy metals and home and may play a protective role in gut inflammation. The mechanisms of induction are unknown but may involve glutathione (GSH) modulation AIM: To study the interplay between GSH level and HO-1 induction in the human intestinal epithelial cell line, DLD-1. METHODS: DLO-1 cells were grown in DMEM with 10 % foetal bovine serum. HO-1 expression was assessed by Western-blotting and GSH levels were determined by the DTNB method. N-acetylcysteine (NAC) was used as a promoter of GSH production and MG132, a proteasome inhibitor, was used to inhibit NF-KB. RESULTS: Incubation of DLD-1 cells with home (50/~M) or cadmium chloride (CdCI2; 10 p.M) caused a rapid decrease in GSH levels starting from 10 min after exposure. Maximal effect of home or CdCI2was seen after lh with GSH level decreasing significantly(P< 0.01) from 23 - 0.5 at baseline to 13 _ 2 and 11 _+ 1.5 nmol/mg protein, respectively. This was followed by HO-1 protein expression detectable from 2h while GSH level returned to baseline. Co-incubating the cells with NAC (10 raM) and home (50/~M) led to a similar GSH depletion (14 -+ 1 nmol/mg protein) and HOot protein expression pattern as with home alone. In contrast, pre-treatment (3h) with NAC prevented the early GSH depletion induced by home and delayed HO-1 expression until 4h. NF-KB inhibition is unlikely to be involved in the NAC effects since MG132 (10 #M) did not modify the HO-1 protein expression induced by home. CONCLUSIONS:GSH depletion may participate in the rapid HD-1 protein induction by HO-1 inducers such as home and CdCI2in human intestinal epithelial cells.

3759 Flise-lnhibitory Protein (FLIP) Inhibits NF-KB Signaling And Is Associated With An Increased Susceptibility To Fas-Medinted Apoptneis In Differentiated HT-29 Cells. Maria Pia Russo, Ryan Balfour Sartor, Christian Jobin, Univ of North Carolina, Chapel Hill, NC Introduction: Signal-induced cell death and proinflammatory gone expression are controlled by an intricate combination of signaling proteins that either block or enhance signal transduction. FLIP has been shown to inhibit Fas Ab-induced apoptosis in lymphocytic cell lines. We have previously shown that IEC differentiation causes an increased susceptibility to Fas Abmediated apoptosis. Aims: To investigate the role of FLIP in Fas signaling to NF-KB and its potential effect on IEC cell death. Methods: Undifferentiated HT-29 (HT-29/p) and methotrexatedifferentiated (HT-29/MTX) cells were used to dissect the Fas signaling pathway leading to

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