- Email: [email protected]
Emergence of perianal fistulae and ulceration in a murine model of IBD: A novel model of perianal Crohn's disease (CD)
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
A-122
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
A-122