- Email: [email protected]
Inflammatory bowel disease in A20-deficient mice
human chromosome 1 to 22 and X, and carried out loss of heterozygosity (LOH) analysis. High frequency of LOH (62.5%) was detected on chromosome 6, and then we investigated it further. Five microsatellite markers were chosen on chromosome 6 and we isolated cosmids including 5 microsatellite markers, respectively.After definition of physical order of 5 microsatellite markers using fluorescent in situ hybridization, we performed LOH analysis and finally. determined commonly deleted region between two microsatellite loci. [CONCLUSION] We conclude that novel tumor suppressor genes associated with colitic cancer exist in chromosome 6.
of colitis. However. the epithelial cell-derived pathogenic antigens have not been identified. The serological analysis of recombinant cDNA expression libraries (SEREX) is a powerful method to define immunogenic antigens that elicit cellular as well as humoral immunity. Since the spontaneous colitis in T cell receptor (TCR) ~x knockout (KO) mice is associated with markedly increasedautoantibody production, this mouse provides a useful model for identifying potential pathogenic self-antigens by the SEREX approach. METHODS:The directional cDNA libraries constructed from freshly isolated colonic epithelial cells of TCRe KO mice were screened by purified immunoglobulin from TCRo~KO mice with colitis. The positive clones were subjected to DNA sequence and suhcloned into pGEX-6P vector to produce GST-fusion proteins.The recombinant (r) proteins were purified using glutathione sepharose4B, followed by using eodotoxin-removal column. Since IL-4 and IL-6 are involved in the pathogenesis of colitis in TOR~ KO mice (Gastroenterology, 1999;116:320), the immunogenic activity of rproteins was screened by the detection of IL-4 and IL-6 productions by ELISA using supematant of the cultured masentedc lymph node (MLN) cells with r-proteins for 3 days. RESULTS: We identified a r-protein Capableof inducing IL-6 production by MLN cells and determined the sequenceof this r-protein as the full-length cDNA (-37 to + 1029) of mouse galectin-4 (64). Aithogh SEREXalso detected a positive clone that was identified to carry the full-length cDNA encoding other galactin family, galectin-3 (G3), this protein could not induce IL-6 production. RNaseprotection assaysshowed that intrapedtoneal injection of 64, but not G3 led to increased I L-6 mRNA expression in MLN of TCRozKO mice. We generatedmonoclonal antibodies specific for mouse 64 and confirmed that G4 is expressed only in the epithelial cells of the colon. CONCLUSION:These Studies raise a possibility that the colonic epithelial cell-derived galectin4 may be a potential pathogenic mediator in the progression and/or perpetuation of chronic colitis in TCRa KO mice.
65,1 Evidence that IL-18 Plays a Protective Role in an in Vivo Inflammatory Model of Epithelial Damage and Repair Basak Coruh, Marco Marini, Brian 0. Weber, Christopher h. Moskaluk, Theresa T. Pizarro, Univ of Virginia, Charlottesville, VA The intestinal epithelium is an active participant in establishing and maintaining mucosal immunity and has the ability to produce a variety of immunoregulatory mediators in response to epithelial activation or injury. We have previously shown that the major source of IL-18 within the gut mucosa is intestinal epithelial cells (IEC). IEC-derived IL-18 is upregulated during chronic intestinal inflammation and in Crohn's disease (CD), there is dramatic shift ot IL-18 expression from IEC to tissue histiocytas (Mq~) and dendritic cell (DC) as severity of disease increases. The aim of the present study was to investigate, in vivo, the role of 11.-18 in mediating mucosal immune responses in an acute inflammatory model of epithelial cell injury and repair. 3-5% DSS in drinking water of IL-18 KO and Cont C57BI6/J mice iN=6/ time point) was administered for 5d followed by a 7d recovery period, after which surviving mice were sac'd. Water intake, hemoccult status and weight were monitored, and colonic tissues collected for histologic assessment and IL-18 immunostainiog. Serum and supesfrom mesenteric lymph node (MLN) (stimulated + / - /.LCD3(1.5 X 10~ cells/ml) were collected for cytokine measurements. 50% mortality was observed in IL-18 KO mice compared to 0% in Cont (5% DSS); all mice succumbing to death did so during the recovery phase. Histologic assessment showed areas of active epithelial regeneration and restitution, but with increased levels of active inflammation (5.1 _+1.1 vs 2.0_+0.5for 3%, p-O.02; 3.7_+0.8 vs 0.8_+0.3 for 5%, p-~0.2) and areas of ulcerations (3.4_+0.7 vs 0.7_+0.3 for 3%, p-
m Emefleace Of Pedanal Fistulae And Ulceration In A Murine Model Of IBD: A Novel Model Of Pedanal Crohn's Disease (CD) Steven M. Cohn, Alda Viddch, Michelle Summy, Christopher A. Moskaluk, Univ of Virginia Health System, Charlottesville, VA; Fabio Cominetli, Univ of Virginia, Charlottesville, VA BACKGROUND:Perianal disease is an important complication of CD. However, until now, no animal models of fistuliziog pedanal IBD have been reported. SAMP1/Yit mice spontaneously develop chronic terminal ileitis with histopathologic features closely resembling human CD (Matsumoto etal., Gut 1998;43:71-8). Significant mucosal lesions develop in the ileum at 10 wks of age, and by age 30 wks nearly 100% of mice show segmental acute and chronic inflammation with transmural involvement, villous blunting, goblet cell and Paneth cell hyperplasia. Crypt abscesses and granuloma formation are observed in some mice. Ileitis is not observed in gnotobiotic SAMPt/Yit mice. METHODS: Multiple inbred sublines of SAMP1Hit mice were developed from the UVA colony, which has been maintained for 11 generations from the original sets of breeding pairs. RESULTS:We observed the simultaneous emergence of fistulizing perlanal disease in all generated sublines. The nearest common ancestor among the affected sublines preceded emergence of disease by 9 generations. The incidence of pedanal disease was 4.8%. Typical lesions developed in young mice with a median age of onset at 7 wks (range 3.6-24 wks) and a female predominance (male:female 1:6.7). Macroecopically, there was loss of fur and ulceration of the perianal skin. Dilated perineal glands, fissuring ulcers, and/or fistulous tracks were frequently observed in affected animals. Histological examination revealed deep ulcerations of perianal skin, rectal ulceration, and fistulous tracks seen in subcutaneous tissue, extending into the rectum. Transmural active and chronic inflammatory infiltrates were observed in the rectum, anus, and subcutaneous tissue. These histological features closely resemble perianal lesions that develop in a subpopulation of human patients with CD. CONCLUSION:To our knowledge, this is the first description of an animal model of periana/CO-like disease. Taken together with previous observations of ileitis in the SAMP1/Yit mouse, these data suggest that distinct environmental or microbial factors leadto the development of fistuliziog perlanaldiseaseand ileitis in a common genetically susceptible host.
654 Leptin-Deficient (Ob/Ob) Mice Are Resistant To Experimental Colitis: Role Of ProInflammatory Cytokines Britta Siegmund, Fabia Gamboni-Robertson, Univ of Colorado Health Science Ctr, Denver, CO; Charles A. Oinarello, Univ of Colorado, Denver, CO; Giamila Fantuzzi, Univ of Colorado Health Science Ctr, Denver, CO Leptin the product of the ob gene, regulates the balance of Thl/Th2 cytokines and modifies T cell immunity. To study the influence of leptin in autoimmune diseasessuch as inflammatory bowel disease (IBD), leptin-deticient ob/ob mice, with or without leptin replacement, or wild type (WT) controls received 3.5% dextran sulfate sodium (DSS) in the ddnloog water for five days and were observed until day ten. Ob/ob mice showed an over 70% reduction of colitis severity (weight loss, stool consistency and bleeding) and colon shortening compared to W'I mice. The absence of colitis in ob/ob mice was accompanied by a marked suppression of IL-18 expression in the epithelial cells of the transversing colon compared to DSS-tad WT mice as evaluated by immunostaining. Leptin replacement, started five days before DSS and continued until the end of DSS exposure, resulted in a significant increase of IL-18 expression and diseaseseverity in both ob/ob and w r mice, whereas almost no IL-1B could be determined in non-DSS exposed mice. Consistent with the immunostaining for IL-16, TNFot, IL-1B and IFN'yconcentrations in the supernatant of total colon culture were markedly suppressed in DSS-fed ob/ob compared to WT mice. Leptin substitution in DSS-tad ob/ob mice led to a significant increase of IL-18, IFN~and TNFofinthe colon culture supernatant, comparable to the concentrations observed in DSS-fed WI mice. Remarkably, IL-lO concentrations were lower in ob/ob and WT DSS-exposedmice compared to non-DSS controls and did not change in the leptin-replaced DSS-fedgroups. As a systemic parameter, Con A-stimulated splenocytes of DSS-fed ob/ob mice showed significantly lower levels of TNF~and IFN~tthanthe DSS-fed WI mice. As leptin replacement converted disease resistance to disease susceptibility, we conclude that leptin represents a crucial link between the endocrine and the immune system which requires further investigation in human IBD. The study was supported by NIH-AI-16614 and DFG SI 749/2-1.
We are grateful to Dr. S. Matsumoto of the Yakult Institute for Microbiological Research in Tokyo, Japan who developed the SAMP1/Yit mouse strain.
657 Inflammatory Bowel Disease in A20-Deficient Mice David L. Boone, Eric G. Lee, Sophia Chai, James P. Lodolce, Shun Libby, Marcia Chien, Suneeta Fernandez, Paula J. Gibson, Averil Ma, Univ of Chicago, Chicago, IL BACKGROUND:The zinc finger protein A2O inhibits NF-kappaB(NF-kB) activation by TNF and other cytokines. To study the function of this protein in vivo we generated mice with a targeted deletion of the A20 gene. Cells from A20 deficient (A2O-/-) mice were unable to negatively regulate TNF-induced NF-kBand A 2 0 - / - mice developed inflammatory bowel disease (IBD). The objective of this study was to characterize the IBD observed in A 2 0 - / - mice and to examine the function of A20 as a regulator of hematopoeitJc JJneagecells in the intestine. METHOD: Lamina propda cells from wild type and A 2 0 - / - mice were isolated for analysis by flow cytometry and intracytoplasmic cytokine staining. Additionally, to determine the specific function of h20 in hematopoeitic cells, these analyses were performed on lamina propda cells isolated from RAG2-/- mice adoptively transferred with wild-type and A 2 0 - / - fetal liver derived hematopoeitic stem cells. RESULTS: The lamina propria of A 2 0 - / - mice contained dramatically increased numbers of activated macrophages and granulocytes. These cells displayed enhanced expression of IL-12 and TNF. Adoptively transferred A 2 0 - / - , but not wild-type, fetal liver cells caused IBD in RAG2-/- recipients. Similar to A 2 0 - / - mice, the lamina propria of A 2 0 - / - hematopoeitic stem cell recipients contained increased numbers of granulocytes and macrophagesthat expressedelevatedTNFand IL-12. In addition, adoptively transferred A 2 0 - / - T lymphocytes isolated from the lamina propria of RAG2-/- recipients expressed increased IFN-gamma compared to wild-type controls. CONCLUSIONS: In the absence of A20, mice develop IBD characterized by increased TNF and IL-12 expressing granulocytes and macrophages. This IBD can occur following adoptive transfer of A 2 0 - / -
655 Colonic Epithelial Cell-Derived Galectin-4 as a Potential Pathogenic Mediator in the Chronic Colitis of TCRo~Knockout Mice Akira Hokama, Yosuke Tanaka, Emiko Mizogucbl, Scott B. Snapper, Atul K. Bhan, Atsushi Mizoguchi, MA Gen Hosp, Boston, MA BACKGROUNDAND AIM: Several studies using experimental colitis models for human inflammatory bowel disease indicate that enteric bacteria are required for the development of colitis. The antigens present in the intestinal epithelial cells may also participate in the pathogenesis
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hematopoeitic cells, demonstratingthat A20 is required for the proper regulation of hematopoeitic lineage ceils in the intestine. These results suggest that disrupted negative regulation of NF-kB in cells of the hematopoeitic lineage can lead to IBD.
Transfection of these clones into the cell lines revealed regions or elements that serve to either enhanceor repressbasaltransactivationof the plgR gene. Band shift assaysof overlapping primers within the core promoter identified eight distinct complexes,the exact locations of which were determined by competition experiments with oligonucleotidesthat contained specific mutations. Many of these complexes appear to be both unique and tissue-specific. An E-box located betweao -71 and -66 was determioed to bind the transcriptional factors USF-1 and 2. An 18-nucleotide insert unique to the human isoform of the gene formed three specific complexes that appear to be formed by novel nuclear factors that have distinct repressor and activator roles. An element located between -122 to -133 strongly resembles the putative binding site for the C/EBPand servesto repressbasalexpression.Another element that weakly resembles a cAMP-responsive element was identified between -136 and -144. Finally, a tissue-specific complex that is similar but distinct from the homeobox proteins Cdx2 and Nkx-2.5were also identified. CONCLUSIONS:In summary, we report the characterization of the human p/gR promoter and the essential role that eight different nuclear complexes have in controlling basal expression of this gene in various cell lines.
658 The Role of Mouse Strain and Thl/Th2 Immune Responses in the al,2Fucosyltrancterase Transgenic Mouse Model of Colitis. Ashley M. Miller, Peter R. Elliott, William Connell, Steven Brown, Paul V. Desmond, Anthony J. F. D'Apice, St Vincent's Hosp, Melbourne Australia Background: BALB/c and C57B1_/6are mouse strains with differing susceptibility to colitis in reported murine models of colitis. Mudne models of mucosal inflammation may be characterised as Thl models, with overproduction of IL-12, IFN~/and TNF~, or Th2 models with overproduction of IL-4. STAT4and STAT6are nuclear transcription proteins that mediateThl (via IL-12 signalling) and Th2 (via IL-4 signalling) responses respectively.Aim: To examine the role of mouse strain and Thl/Th2 immune responsesin the human al,2-fucosyitransferase (hFUT1) transgenic mouse model of colitis. Methods: We have previously reported that mice transgenic for hFUT1 spontaneouslydevelop colitis [Gastroenterol May 2000 A687.]. hFUT1 transgenic mice were originally generatedon a mixed genetic backgroundof CBA and C57BI/ 6. hFUT1 transgenic mice were backcrossed onto BALB/c and C57BL/6 mouse strains, and onto STAT4 and STAT6 knockout mice. STAT genotype was assessed by PCR. Colitis was confirmed histologically. Results: The incidence of colitis in the original hEUT1 transgenic mice was 22%. The incidenceof colitis progressivelyincreasedin eachgenerationof transgenic mice backcrossedonto a BALB/c background.The incidenceof colitis was 27% at backcross generation N2, 46% at N3, 52% at N4, 63% at N5, and 64% at N6. Colitis failed to develop when the transgenewas backcrossedonto a C57B!d6 background,hFUT1+ STAT4or STAT6 knockout mice were generatedat BALB/cN3 generation.Sevenof 14 (50%) of hFUT1+ STAT6 knockout mice developedcolitis, comparableto control hFUT1+ mice backcrossedto a BALB/ c N3 generation (46%). No hFUT1+ STAT;4 knockout mice developed colitis. Conclusion: Developmentof colitis in hFUT1 transgenic mice requires an intact Thl response. In addition, other genetic influences on colitis developmentare apparentfrom the effects of backcrossing onto BALB/c and 057BIE6 mouse strains.
661 HNF-I~ and HNF-1,8 Regulate Transcriptional Activation of the Rat Liver Fatty Acid Binding Protein Geue in Liver, Intestine, and Kideey by Otstioct Interactions with Cdx, C/EBP, GATA, HNF-3, and HNF-4 Family Transcription Factors Joyce K. Divine, Scan P. McCaul, Theodore C. Simon, Washington Univ Sch of Medicine, St. Louis, MO Members of the Cdx, C/EBP, GATA, HNF-1, HNF-3, and HNF-4 transcription factor families comprise a transcriptional regulatory network critical both for endoderm specificationand for the expression of genes involved in energy metabolism. Binding sites for all these factors were identified in the proximal promoter of the rat liver fatty acid binding protein gene (Fabp/). These sites were located in a compact sequence spanning Fabp/nucieotides -178 to -55 relative to the start site of transcdption. Transient transfeetion assays were performed in CaCo-2cells, HepG2cells, and LLC-PK~cells with a transgeneconstructedfrom Fabplnucleotides -596 to +21. Members of each of these transcription factor families trans-activated the ,Cab#/transgene,and trans-activation by each factor was dependenton an intact cognate binding sits(s). Fabp/trans-activation by a combination of factors from all the transcription factor families resulted in a more than ten-fold greater transgene activity than the sum of activation by the individoa~factors. Functionalsy~ergy betweentranscription factors may thus be the most critical determinantof gene expressionin a particular cell. In particular, we found that HNF-1 accounted for half the total synergistic activity of the combined factors. The importance of HNF-1 in Fabp/expression was determined by comparing the expression of the native Fabpltransgene in mice to that of an identical transgene with a specific sequence mutation that preventedHNF-1 binding. The nativetransgene is active in the intestinal epithelium, hepatocytes,and renal proximal tubular epithilium, but the same transgene without a functional HNF-1 site was silent in all these tissues. In contrast, transgenes with mutations in HNF-4, GATA, and Cdx binding sites were active in the intestinal epithelium. Furthermore, specific synergy was noted in cell transfections between HNF-la or HNF-1/3 and the other family factors. HNF-la but not HNF-1/~exhibited synergy with other factors to activate Fabp/ in CaCo-2 and HepG2 cells, with the reverse observed in LLC-PK1 cells. HNF-la is the predominant species in the gut and liver, while HNF-1/3is the predominant species in kidney. These experiments provide evidencethat distinct and tissue-specific interactions of HNF-1~ and HNF-I,8 with other endodermal transcriptional regulators are critical for defining gene expression in vivo.
659 Physical Interaction Between GATA-5 and HNF-la Results in Synergistic Activation of the Human Lactase-Phlorizin Hydrolase Promoter Herbert M. Van Wering, Inge L. Huibregtse, Richard J. Grand, Stephen D. Krasinski, New England Medical Ctr, Boston, MA GATA-5and HNF-la transcription factors are both expressedin intestinal epithelium and have been shown to activate intestinal gene promoters. Due to the presenceand configuration of binding sites for GATAand HNF-1 in the 5'-flanking sequenceof the human lactase-phlorizin hydrolase (hLPH) gene, we hypothesizedthat GATA-5 and HNF-I~ interact with each other to activate hLPH gene expression. Co-transfection into Caco-2 cells of an hLPH promoterhuman growth hormone reporter construct containing 118 bp of hLPH 5'-flanking region with expression vectors for GATA-5 and HNF-I,~ together resulted in a higher activation of the hLPH promoterthan the sum of the activationof eachfactor alone,demonstratingfunctional co-operativity. Mutational analysisof the GATAand HNF-1 binding sites in the hLPH promoter revealedthat the functional co-operativity is dependenton an intact HNF-t binding site, but not on intact GATAbinding sites. Physicalinteraction betweenGATA-5and HNF-la was shown in vitro by GST pull-down assays.Mutation and deletion analysisof GATA-5demonstratedthat the regions responsible for GATA-5/HNF-I~ and GATA-5/DNAinteractions are localized in close proximity to each other in domains that include the carboxy-terminal zinc finger and basic region; these interactions did not localize to the amino-terminal zinc-finger. However, a GATA-5 mutant containing specific amino acid substitutions at 256-258 (TTT to SSS) was identified that disrupts DNA-binding but not physical interaction with HNF-la. This protein demonstrated complete GATA-5/HNF-I~ functional co-operativity in co-transfection assays, further supporting the hypothesis that GATA-5 interaction with DNA is not necessary for synergistic activation of the hLPH promoter. CONCLUSIONS:(1) Physical interaction between GATA-5 and HNF-la results in synergistic activation of the hLPH promoter. (2) Functional co-operativity requires binding of HNF-I~ to the HNF-1 cis-element in the bLPH promoter, but does not require binding of GATA-5to the GATAcis-element. (3) These data identify an essential role for HNF-le in the expression of the LPH gene and involvement of GATA-5for synergistic activation.
662 Luminal Bile Acids Influence the Developmental Expression of Ileal Bile Acid Binding Protein by Activation of the Bile Acid Receptor FXR Sandy T. Hwang, David Moore, Susan J. Henning, 8aylor Coil of Medicine, Houston, TX BACKGROUND/OBJECTIVES:In the rat, there is a dramatic increase in ileal bile acid binding protein (IBABP) mRNA and protein levels during the third postnatal week. Developmental changes in bile acid metabolism suggest that there are increased luminal bile acids at this time. Moreover, the IBABP promoter contains the response element (IR-1) for the newly discovered bile acid receptor, FXR. However, no prior studies have examined the influence of luminal bile acids on the ontogenic expressionof IBABP.Thus, our aims were to determine the role of bile acids on IBABP expression in the suckling rat and whether FXR mediates these effects. METHODS:Animals were placed into one of the following groups- untreated, vehicle gavage,dexamethasone(DEX),taurocholategavage,and DEX + taumcholate.Animals were treated on days 13-15. Ileum was collected on day 16 for Northern and Western blot analyses. Next, nuclear extracts were obtained from mucosal scrapings of distal ileum from different aged rats. Electrophoreticmobility shift assayswere performedusing nuclearproteins incubated with a radiolabelledIR-1 oligonucleotide. Cold competition was done with 100 and lO00-fold molar excess of both unlabelled IR-1 and mutated IR-I. Supershiff assays were performed using both a FXRantibody as well as a nonspecificantibody. RESULTS:Taurocholate gavagesignificantlyelevatedIBABPmRNAand protein levelsin day 16 animalsto approximately 30% and 60% of adult levels, respectively.These levels were significantly higher than those seen with DEX,and no interaction between bile acid gavageand DEX was detected. Gelshifts with nuclear extracts from adult rat ileum revealeda DNA-protein complex which represented FXR binding to IR-1. This complex was competed away with excesscold IR-1 but not mutated IR-l,and was supershifted by FXR antibody but not nonspecific antibody. The FXR DNA binding activity was detected in ileal nuclear extracts from rats at ages in which there is normally increasedexpressionof IBABP(days 22 and 40) but was absent at an age in which there is no IBABP (day 12). CONCLUSION:Bile acids may play a role in the normal developmental expression of IBABP, and this effect appears to be mediated by FXR directly binding to the IR-1 of the IBABPgene. This work was supported by NIH grant ,~ KO8 DK-02550 and P3O HD-27823.
660 Multiple Transcription Factors in the 5 -Flanking Region of the Human Polymeric IOA Receptor (plgR) Controls its Basal Expression. Sergio R. Solorzano-Vargas,CA State Univ Northridge, Los Angeles, CA; Jiatang Wang, Lingling Jiang, Tsai V. Hugh, Ontiveros O. Luis, Vazir A. Mukta, Aguilera J. Renalto, Martin G. Martin, UCLA, Los Angeles, CA BACKGROUND:The polymeric Ig receptor (plgR) is a critical component of the mucosal immune system and is expressed in the small intestine. Expression of plgR is regulated at the level of transcription by various hormones and cytokines. Expressionof the gene is also developmentallyregulated.The control of basal expressionby the immediatepromoter region has not beenthoroughly evaluated.AIMS: To elucidatethe core promoter region of the human plgR gene. METHODS:Chimericclonesthat containedvarious regions of the promoter located just up-stream of luciferase were developed and transiently transfected into Caco2, HT-29 and HepG2cell lines. DNase I footprinting and DNA binding assayswere used in combination to further define the critical elements. RESULTS:Expression of chimeric promoter-reporter constructs was supported in all cell lines, and the core promoter was located between-210 and +76from the start of transcription. DNasel footprint analysis revealed a large complex within the core promoter region located between -46 and -190. Site directed mutagenesis of the core promoter was performed in 10 bp increments, and 20 clones were produced.
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of colitis. However. the epithelial cell-derived pathogenic antigens have not been identified. The serological analysis of recombinant cDNA expression libraries (SEREX) is a powerful method to define immunogenic antigens that elicit cellular as well as humoral immunity. Since the spontaneous colitis in T cell receptor (TCR) ~x knockout (KO) mice is associated with markedly increasedautoantibody production, this mouse provides a useful model for identifying potential pathogenic self-antigens by the SEREX approach. METHODS:The directional cDNA libraries constructed from freshly isolated colonic epithelial cells of TCRe KO mice were screened by purified immunoglobulin from TCRo~KO mice with colitis. The positive clones were subjected to DNA sequence and suhcloned into pGEX-6P vector to produce GST-fusion proteins.The recombinant (r) proteins were purified using glutathione sepharose4B, followed by using eodotoxin-removal column. Since IL-4 and IL-6 are involved in the pathogenesis of colitis in TOR~ KO mice (Gastroenterology, 1999;116:320), the immunogenic activity of rproteins was screened by the detection of IL-4 and IL-6 productions by ELISA using supematant of the cultured masentedc lymph node (MLN) cells with r-proteins for 3 days. RESULTS: We identified a r-protein Capableof inducing IL-6 production by MLN cells and determined the sequenceof this r-protein as the full-length cDNA (-37 to + 1029) of mouse galectin-4 (64). Aithogh SEREXalso detected a positive clone that was identified to carry the full-length cDNA encoding other galactin family, galectin-3 (G3), this protein could not induce IL-6 production. RNaseprotection assaysshowed that intrapedtoneal injection of 64, but not G3 led to increased I L-6 mRNA expression in MLN of TCRozKO mice. We generatedmonoclonal antibodies specific for mouse 64 and confirmed that G4 is expressed only in the epithelial cells of the colon. CONCLUSION:These Studies raise a possibility that the colonic epithelial cell-derived galectin4 may be a potential pathogenic mediator in the progression and/or perpetuation of chronic colitis in TCRa KO mice.
65,1 Evidence that IL-18 Plays a Protective Role in an in Vivo Inflammatory Model of Epithelial Damage and Repair Basak Coruh, Marco Marini, Brian 0. Weber, Christopher h. Moskaluk, Theresa T. Pizarro, Univ of Virginia, Charlottesville, VA The intestinal epithelium is an active participant in establishing and maintaining mucosal immunity and has the ability to produce a variety of immunoregulatory mediators in response to epithelial activation or injury. We have previously shown that the major source of IL-18 within the gut mucosa is intestinal epithelial cells (IEC). IEC-derived IL-18 is upregulated during chronic intestinal inflammation and in Crohn's disease (CD), there is dramatic shift ot IL-18 expression from IEC to tissue histiocytas (Mq~) and dendritic cell (DC) as severity of disease increases. The aim of the present study was to investigate, in vivo, the role of 11.-18 in mediating mucosal immune responses in an acute inflammatory model of epithelial cell injury and repair. 3-5% DSS in drinking water of IL-18 KO and Cont C57BI6/J mice iN=6/ time point) was administered for 5d followed by a 7d recovery period, after which surviving mice were sac'd. Water intake, hemoccult status and weight were monitored, and colonic tissues collected for histologic assessment and IL-18 immunostainiog. Serum and supesfrom mesenteric lymph node (MLN) (stimulated + / - /.LCD3(1.5 X 10~ cells/ml) were collected for cytokine measurements. 50% mortality was observed in IL-18 KO mice compared to 0% in Cont (5% DSS); all mice succumbing to death did so during the recovery phase. Histologic assessment showed areas of active epithelial regeneration and restitution, but with increased levels of active inflammation (5.1 _+1.1 vs 2.0_+0.5for 3%, p-O.02; 3.7_+0.8 vs 0.8_+0.3 for 5%, p-~0.2) and areas of ulcerations (3.4_+0.7 vs 0.7_+0.3 for 3%, p-
m Emefleace Of Pedanal Fistulae And Ulceration In A Murine Model Of IBD: A Novel Model Of Pedanal Crohn's Disease (CD) Steven M. Cohn, Alda Viddch, Michelle Summy, Christopher A. Moskaluk, Univ of Virginia Health System, Charlottesville, VA; Fabio Cominetli, Univ of Virginia, Charlottesville, VA BACKGROUND:Perianal disease is an important complication of CD. However, until now, no animal models of fistuliziog pedanal IBD have been reported. SAMP1/Yit mice spontaneously develop chronic terminal ileitis with histopathologic features closely resembling human CD (Matsumoto etal., Gut 1998;43:71-8). Significant mucosal lesions develop in the ileum at 10 wks of age, and by age 30 wks nearly 100% of mice show segmental acute and chronic inflammation with transmural involvement, villous blunting, goblet cell and Paneth cell hyperplasia. Crypt abscesses and granuloma formation are observed in some mice. Ileitis is not observed in gnotobiotic SAMPt/Yit mice. METHODS: Multiple inbred sublines of SAMP1Hit mice were developed from the UVA colony, which has been maintained for 11 generations from the original sets of breeding pairs. RESULTS:We observed the simultaneous emergence of fistulizing perlanal disease in all generated sublines. The nearest common ancestor among the affected sublines preceded emergence of disease by 9 generations. The incidence of pedanal disease was 4.8%. Typical lesions developed in young mice with a median age of onset at 7 wks (range 3.6-24 wks) and a female predominance (male:female 1:6.7). Macroecopically, there was loss of fur and ulceration of the perianal skin. Dilated perineal glands, fissuring ulcers, and/or fistulous tracks were frequently observed in affected animals. Histological examination revealed deep ulcerations of perianal skin, rectal ulceration, and fistulous tracks seen in subcutaneous tissue, extending into the rectum. Transmural active and chronic inflammatory infiltrates were observed in the rectum, anus, and subcutaneous tissue. These histological features closely resemble perianal lesions that develop in a subpopulation of human patients with CD. CONCLUSION:To our knowledge, this is the first description of an animal model of periana/CO-like disease. Taken together with previous observations of ileitis in the SAMP1/Yit mouse, these data suggest that distinct environmental or microbial factors leadto the development of fistuliziog perlanaldiseaseand ileitis in a common genetically susceptible host.
654 Leptin-Deficient (Ob/Ob) Mice Are Resistant To Experimental Colitis: Role Of ProInflammatory Cytokines Britta Siegmund, Fabia Gamboni-Robertson, Univ of Colorado Health Science Ctr, Denver, CO; Charles A. Oinarello, Univ of Colorado, Denver, CO; Giamila Fantuzzi, Univ of Colorado Health Science Ctr, Denver, CO Leptin the product of the ob gene, regulates the balance of Thl/Th2 cytokines and modifies T cell immunity. To study the influence of leptin in autoimmune diseasessuch as inflammatory bowel disease (IBD), leptin-deticient ob/ob mice, with or without leptin replacement, or wild type (WT) controls received 3.5% dextran sulfate sodium (DSS) in the ddnloog water for five days and were observed until day ten. Ob/ob mice showed an over 70% reduction of colitis severity (weight loss, stool consistency and bleeding) and colon shortening compared to W'I mice. The absence of colitis in ob/ob mice was accompanied by a marked suppression of IL-18 expression in the epithelial cells of the transversing colon compared to DSS-tad WT mice as evaluated by immunostaining. Leptin replacement, started five days before DSS and continued until the end of DSS exposure, resulted in a significant increase of IL-18 expression and diseaseseverity in both ob/ob and w r mice, whereas almost no IL-1B could be determined in non-DSS exposed mice. Consistent with the immunostaining for IL-16, TNFot, IL-1B and IFN'yconcentrations in the supernatant of total colon culture were markedly suppressed in DSS-fed ob/ob compared to WT mice. Leptin substitution in DSS-tad ob/ob mice led to a significant increase of IL-18, IFN~and TNFofinthe colon culture supernatant, comparable to the concentrations observed in DSS-fed WI mice. Remarkably, IL-lO concentrations were lower in ob/ob and WT DSS-exposedmice compared to non-DSS controls and did not change in the leptin-replaced DSS-fedgroups. As a systemic parameter, Con A-stimulated splenocytes of DSS-fed ob/ob mice showed significantly lower levels of TNF~and IFN~tthanthe DSS-fed WI mice. As leptin replacement converted disease resistance to disease susceptibility, we conclude that leptin represents a crucial link between the endocrine and the immune system which requires further investigation in human IBD. The study was supported by NIH-AI-16614 and DFG SI 749/2-1.
We are grateful to Dr. S. Matsumoto of the Yakult Institute for Microbiological Research in Tokyo, Japan who developed the SAMP1/Yit mouse strain.
657 Inflammatory Bowel Disease in A20-Deficient Mice David L. Boone, Eric G. Lee, Sophia Chai, James P. Lodolce, Shun Libby, Marcia Chien, Suneeta Fernandez, Paula J. Gibson, Averil Ma, Univ of Chicago, Chicago, IL BACKGROUND:The zinc finger protein A2O inhibits NF-kappaB(NF-kB) activation by TNF and other cytokines. To study the function of this protein in vivo we generated mice with a targeted deletion of the A20 gene. Cells from A20 deficient (A2O-/-) mice were unable to negatively regulate TNF-induced NF-kBand A 2 0 - / - mice developed inflammatory bowel disease (IBD). The objective of this study was to characterize the IBD observed in A 2 0 - / - mice and to examine the function of A20 as a regulator of hematopoeitJc JJneagecells in the intestine. METHOD: Lamina propda cells from wild type and A 2 0 - / - mice were isolated for analysis by flow cytometry and intracytoplasmic cytokine staining. Additionally, to determine the specific function of h20 in hematopoeitic cells, these analyses were performed on lamina propda cells isolated from RAG2-/- mice adoptively transferred with wild-type and A 2 0 - / - fetal liver derived hematopoeitic stem cells. RESULTS: The lamina propria of A 2 0 - / - mice contained dramatically increased numbers of activated macrophages and granulocytes. These cells displayed enhanced expression of IL-12 and TNF. Adoptively transferred A 2 0 - / - , but not wild-type, fetal liver cells caused IBD in RAG2-/- recipients. Similar to A 2 0 - / - mice, the lamina propria of A 2 0 - / - hematopoeitic stem cell recipients contained increased numbers of granulocytes and macrophagesthat expressedelevatedTNFand IL-12. In addition, adoptively transferred A 2 0 - / - T lymphocytes isolated from the lamina propria of RAG2-/- recipients expressed increased IFN-gamma compared to wild-type controls. CONCLUSIONS: In the absence of A20, mice develop IBD characterized by increased TNF and IL-12 expressing granulocytes and macrophages. This IBD can occur following adoptive transfer of A 2 0 - / -
655 Colonic Epithelial Cell-Derived Galectin-4 as a Potential Pathogenic Mediator in the Chronic Colitis of TCRo~Knockout Mice Akira Hokama, Yosuke Tanaka, Emiko Mizogucbl, Scott B. Snapper, Atul K. Bhan, Atsushi Mizoguchi, MA Gen Hosp, Boston, MA BACKGROUNDAND AIM: Several studies using experimental colitis models for human inflammatory bowel disease indicate that enteric bacteria are required for the development of colitis. The antigens present in the intestinal epithelial cells may also participate in the pathogenesis
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hematopoeitic cells, demonstratingthat A20 is required for the proper regulation of hematopoeitic lineage ceils in the intestine. These results suggest that disrupted negative regulation of NF-kB in cells of the hematopoeitic lineage can lead to IBD.
Transfection of these clones into the cell lines revealed regions or elements that serve to either enhanceor repressbasaltransactivationof the plgR gene. Band shift assaysof overlapping primers within the core promoter identified eight distinct complexes,the exact locations of which were determined by competition experiments with oligonucleotidesthat contained specific mutations. Many of these complexes appear to be both unique and tissue-specific. An E-box located betweao -71 and -66 was determioed to bind the transcriptional factors USF-1 and 2. An 18-nucleotide insert unique to the human isoform of the gene formed three specific complexes that appear to be formed by novel nuclear factors that have distinct repressor and activator roles. An element located between -122 to -133 strongly resembles the putative binding site for the C/EBPand servesto repressbasalexpression.Another element that weakly resembles a cAMP-responsive element was identified between -136 and -144. Finally, a tissue-specific complex that is similar but distinct from the homeobox proteins Cdx2 and Nkx-2.5were also identified. CONCLUSIONS:In summary, we report the characterization of the human p/gR promoter and the essential role that eight different nuclear complexes have in controlling basal expression of this gene in various cell lines.
658 The Role of Mouse Strain and Thl/Th2 Immune Responses in the al,2Fucosyltrancterase Transgenic Mouse Model of Colitis. Ashley M. Miller, Peter R. Elliott, William Connell, Steven Brown, Paul V. Desmond, Anthony J. F. D'Apice, St Vincent's Hosp, Melbourne Australia Background: BALB/c and C57B1_/6are mouse strains with differing susceptibility to colitis in reported murine models of colitis. Mudne models of mucosal inflammation may be characterised as Thl models, with overproduction of IL-12, IFN~/and TNF~, or Th2 models with overproduction of IL-4. STAT4and STAT6are nuclear transcription proteins that mediateThl (via IL-12 signalling) and Th2 (via IL-4 signalling) responses respectively.Aim: To examine the role of mouse strain and Thl/Th2 immune responsesin the human al,2-fucosyitransferase (hFUT1) transgenic mouse model of colitis. Methods: We have previously reported that mice transgenic for hFUT1 spontaneouslydevelop colitis [Gastroenterol May 2000 A687.]. hFUT1 transgenic mice were originally generatedon a mixed genetic backgroundof CBA and C57BI/ 6. hFUT1 transgenic mice were backcrossed onto BALB/c and C57BL/6 mouse strains, and onto STAT4 and STAT6 knockout mice. STAT genotype was assessed by PCR. Colitis was confirmed histologically. Results: The incidence of colitis in the original hEUT1 transgenic mice was 22%. The incidenceof colitis progressivelyincreasedin eachgenerationof transgenic mice backcrossedonto a BALB/c background.The incidenceof colitis was 27% at backcross generation N2, 46% at N3, 52% at N4, 63% at N5, and 64% at N6. Colitis failed to develop when the transgenewas backcrossedonto a C57B!d6 background,hFUT1+ STAT4or STAT6 knockout mice were generatedat BALB/cN3 generation.Sevenof 14 (50%) of hFUT1+ STAT6 knockout mice developedcolitis, comparableto control hFUT1+ mice backcrossedto a BALB/ c N3 generation (46%). No hFUT1+ STAT;4 knockout mice developed colitis. Conclusion: Developmentof colitis in hFUT1 transgenic mice requires an intact Thl response. In addition, other genetic influences on colitis developmentare apparentfrom the effects of backcrossing onto BALB/c and 057BIE6 mouse strains.
661 HNF-I~ and HNF-1,8 Regulate Transcriptional Activation of the Rat Liver Fatty Acid Binding Protein Geue in Liver, Intestine, and Kideey by Otstioct Interactions with Cdx, C/EBP, GATA, HNF-3, and HNF-4 Family Transcription Factors Joyce K. Divine, Scan P. McCaul, Theodore C. Simon, Washington Univ Sch of Medicine, St. Louis, MO Members of the Cdx, C/EBP, GATA, HNF-1, HNF-3, and HNF-4 transcription factor families comprise a transcriptional regulatory network critical both for endoderm specificationand for the expression of genes involved in energy metabolism. Binding sites for all these factors were identified in the proximal promoter of the rat liver fatty acid binding protein gene (Fabp/). These sites were located in a compact sequence spanning Fabp/nucieotides -178 to -55 relative to the start site of transcdption. Transient transfeetion assays were performed in CaCo-2cells, HepG2cells, and LLC-PK~cells with a transgeneconstructedfrom Fabplnucleotides -596 to +21. Members of each of these transcription factor families trans-activated the ,Cab#/transgene,and trans-activation by each factor was dependenton an intact cognate binding sits(s). Fabp/trans-activation by a combination of factors from all the transcription factor families resulted in a more than ten-fold greater transgene activity than the sum of activation by the individoa~factors. Functionalsy~ergy betweentranscription factors may thus be the most critical determinantof gene expressionin a particular cell. In particular, we found that HNF-1 accounted for half the total synergistic activity of the combined factors. The importance of HNF-1 in Fabp/expression was determined by comparing the expression of the native Fabpltransgene in mice to that of an identical transgene with a specific sequence mutation that preventedHNF-1 binding. The nativetransgene is active in the intestinal epithelium, hepatocytes,and renal proximal tubular epithilium, but the same transgene without a functional HNF-1 site was silent in all these tissues. In contrast, transgenes with mutations in HNF-4, GATA, and Cdx binding sites were active in the intestinal epithelium. Furthermore, specific synergy was noted in cell transfections between HNF-la or HNF-1/3 and the other family factors. HNF-la but not HNF-1/~exhibited synergy with other factors to activate Fabp/ in CaCo-2 and HepG2 cells, with the reverse observed in LLC-PK1 cells. HNF-la is the predominant species in the gut and liver, while HNF-1/3is the predominant species in kidney. These experiments provide evidencethat distinct and tissue-specific interactions of HNF-1~ and HNF-I,8 with other endodermal transcriptional regulators are critical for defining gene expression in vivo.
659 Physical Interaction Between GATA-5 and HNF-la Results in Synergistic Activation of the Human Lactase-Phlorizin Hydrolase Promoter Herbert M. Van Wering, Inge L. Huibregtse, Richard J. Grand, Stephen D. Krasinski, New England Medical Ctr, Boston, MA GATA-5and HNF-la transcription factors are both expressedin intestinal epithelium and have been shown to activate intestinal gene promoters. Due to the presenceand configuration of binding sites for GATAand HNF-1 in the 5'-flanking sequenceof the human lactase-phlorizin hydrolase (hLPH) gene, we hypothesizedthat GATA-5 and HNF-I~ interact with each other to activate hLPH gene expression. Co-transfection into Caco-2 cells of an hLPH promoterhuman growth hormone reporter construct containing 118 bp of hLPH 5'-flanking region with expression vectors for GATA-5 and HNF-I,~ together resulted in a higher activation of the hLPH promoterthan the sum of the activationof eachfactor alone,demonstratingfunctional co-operativity. Mutational analysisof the GATAand HNF-1 binding sites in the hLPH promoter revealedthat the functional co-operativity is dependenton an intact HNF-t binding site, but not on intact GATAbinding sites. Physicalinteraction betweenGATA-5and HNF-la was shown in vitro by GST pull-down assays.Mutation and deletion analysisof GATA-5demonstratedthat the regions responsible for GATA-5/HNF-I~ and GATA-5/DNAinteractions are localized in close proximity to each other in domains that include the carboxy-terminal zinc finger and basic region; these interactions did not localize to the amino-terminal zinc-finger. However, a GATA-5 mutant containing specific amino acid substitutions at 256-258 (TTT to SSS) was identified that disrupts DNA-binding but not physical interaction with HNF-la. This protein demonstrated complete GATA-5/HNF-I~ functional co-operativity in co-transfection assays, further supporting the hypothesis that GATA-5 interaction with DNA is not necessary for synergistic activation of the hLPH promoter. CONCLUSIONS:(1) Physical interaction between GATA-5 and HNF-la results in synergistic activation of the hLPH promoter. (2) Functional co-operativity requires binding of HNF-I~ to the HNF-1 cis-element in the bLPH promoter, but does not require binding of GATA-5to the GATAcis-element. (3) These data identify an essential role for HNF-le in the expression of the LPH gene and involvement of GATA-5for synergistic activation.
662 Luminal Bile Acids Influence the Developmental Expression of Ileal Bile Acid Binding Protein by Activation of the Bile Acid Receptor FXR Sandy T. Hwang, David Moore, Susan J. Henning, 8aylor Coil of Medicine, Houston, TX BACKGROUND/OBJECTIVES:In the rat, there is a dramatic increase in ileal bile acid binding protein (IBABP) mRNA and protein levels during the third postnatal week. Developmental changes in bile acid metabolism suggest that there are increased luminal bile acids at this time. Moreover, the IBABP promoter contains the response element (IR-1) for the newly discovered bile acid receptor, FXR. However, no prior studies have examined the influence of luminal bile acids on the ontogenic expressionof IBABP.Thus, our aims were to determine the role of bile acids on IBABP expression in the suckling rat and whether FXR mediates these effects. METHODS:Animals were placed into one of the following groups- untreated, vehicle gavage,dexamethasone(DEX),taurocholategavage,and DEX + taumcholate.Animals were treated on days 13-15. Ileum was collected on day 16 for Northern and Western blot analyses. Next, nuclear extracts were obtained from mucosal scrapings of distal ileum from different aged rats. Electrophoreticmobility shift assayswere performedusing nuclearproteins incubated with a radiolabelledIR-1 oligonucleotide. Cold competition was done with 100 and lO00-fold molar excess of both unlabelled IR-1 and mutated IR-I. Supershiff assays were performed using both a FXRantibody as well as a nonspecificantibody. RESULTS:Taurocholate gavagesignificantlyelevatedIBABPmRNAand protein levelsin day 16 animalsto approximately 30% and 60% of adult levels, respectively.These levels were significantly higher than those seen with DEX,and no interaction between bile acid gavageand DEX was detected. Gelshifts with nuclear extracts from adult rat ileum revealeda DNA-protein complex which represented FXR binding to IR-1. This complex was competed away with excesscold IR-1 but not mutated IR-l,and was supershifted by FXR antibody but not nonspecific antibody. The FXR DNA binding activity was detected in ileal nuclear extracts from rats at ages in which there is normally increasedexpressionof IBABP(days 22 and 40) but was absent at an age in which there is no IBABP (day 12). CONCLUSION:Bile acids may play a role in the normal developmental expression of IBABP, and this effect appears to be mediated by FXR directly binding to the IR-1 of the IBABPgene. This work was supported by NIH grant ,~ KO8 DK-02550 and P3O HD-27823.
660 Multiple Transcription Factors in the 5 -Flanking Region of the Human Polymeric IOA Receptor (plgR) Controls its Basal Expression. Sergio R. Solorzano-Vargas,CA State Univ Northridge, Los Angeles, CA; Jiatang Wang, Lingling Jiang, Tsai V. Hugh, Ontiveros O. Luis, Vazir A. Mukta, Aguilera J. Renalto, Martin G. Martin, UCLA, Los Angeles, CA BACKGROUND:The polymeric Ig receptor (plgR) is a critical component of the mucosal immune system and is expressed in the small intestine. Expression of plgR is regulated at the level of transcription by various hormones and cytokines. Expressionof the gene is also developmentallyregulated.The control of basal expressionby the immediatepromoter region has not beenthoroughly evaluated.AIMS: To elucidatethe core promoter region of the human plgR gene. METHODS:Chimericclonesthat containedvarious regions of the promoter located just up-stream of luciferase were developed and transiently transfected into Caco2, HT-29 and HepG2cell lines. DNase I footprinting and DNA binding assayswere used in combination to further define the critical elements. RESULTS:Expression of chimeric promoter-reporter constructs was supported in all cell lines, and the core promoter was located between-210 and +76from the start of transcription. DNasel footprint analysis revealed a large complex within the core promoter region located between -46 and -190. Site directed mutagenesis of the core promoter was performed in 10 bp increments, and 20 clones were produced.
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