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Immune modulation by intestinal epithelial cell differentiation
April 1995
• VIDEOENDOSCOPIC OBSERVATION OF ET-I INDUCED MUCOSAL ISCHEMIA: EFFECT OF AN ETA RECEPTOR SELECTIVE ANTAGONIST (FR139317). M. Hosokawa*, M. Tominaga÷, H. Nakamura ÷, T. Taniguchi*, S. Ueda÷, H. Tsukada*, M. Okuma ÷, M. Sakai + Kyoto Police Hospital*, Internal Medicine I. Kyoto University, Kyoto 606-01 Japan" Endothelin (ET) has been originally identified as a potent vasoconstrictive peptide in cultured media of porcine aortic endothelial cells. Recently, we reported that ET-1 enhanced both CI- secretion and muscle tension in rat colon. In this study, we determined how ET-1 induced mucosal damage in rat distal colon by real time observation with new videoendoscopic system (TOSHIBA TRE-300D, Japan). Furthermore we measured mucosal blood flow with laser doppler flowmetry (LDF) during control and condition period for ET-1. Methods: Carefully we washed distal colon of ether-anesthetized male rats with warm water and inserted videoendoscope. We kept observing colonic mucosa while and after sprinkling of ET-1 from biopsy channel with teflon tube. Mucosal blood flow was measured with LDF technique during endoscopy with flow probe inserted down the biopsy channel of endoscope. Results: Submucosal arteriole and venule contracted immediately after ET-1 administration (80 nmol/kg) and mueosal surface turned pale by the ischemic events. Approximately 50 rain took for both vessels to return normal in their diameter and color, showing blood reperfusion to mucosa. At this time small bleeding spots also appeared, which seemed to be extravasation localized inside the mucosa. Both LDF response to ET-I and histological findings were consistent with the videoendoscopic findings. All ET-l-induced changes were inhibited by pre-sprinkling of FR139317 (800 nmol/kg). Conclusion: Our results show that the videoendoscopes have so high resolution that we can detect mucosal ischemia induced by ET-1. FRi39317 inhibits effectively the ET-l-induced mucosal ischemia. FR139317 may provide a useful new therapy for inflammatory bowel diseases because local high concentration of ET-I is reportedly associated with this type of diseases. The new videoendoscope system was certified to be of great use for basic research of gastrointestinal pathophysiology.
• PURIFICATION AND CHARACTERIZATION OF AN ACIDINHIBITORY PROTEIN FROM HELICOBACTER PYLORI. L.L; Huang, D.R. Cave, and A.V. Kane. St. Elizabeth's Medical Center, and New England Medical Center, Tufts University Medical School, Boston, MA. Previous experiments have shown there are two factors produced by Helicobacter pylori that inhibit acid secretion from isolated gastric glands obtained from several mammaliaa species. Acid inhibitory factor 1 (AWl) is ddstroyed by pronase and heat. AIF2 is heat and pronase resistant and is of MW < 2kDa. This study describes the purification and characterization of AIF1 fromH, pylori. Six H. pylon isolates were cultured and tested for the production of AWl using the inhibition of uptake of 14C-amiunpyrine by secretogogue stimulated isolated rabbit gastric glands. The activity of AWl was defined as one unit of AWl being the amount required to inhibit the uptake of 14Caminopyrine by 50%. All isolates examined had inhibitory activity, in which the activity of AWl ranged from 1-65%. The most inhibitory isolate was then cultured under microaerophilic conditions in a fermenter, using brucella broth supplemented with 0.2% cyelodextrin to provide 10 L batches. The bacterial pellet was sonicated, and the supematant was then subjected to filtration through an Amicon membrane (MW cut off 10 kDa). The activity of AIFl remained in the retentate and AIF2 was in the filtrate. The retentate was then sequmtially subjected to hydroxyapatite, hydrephobic and size exclusion chromatography. Fractionswere tested for acid inhibitory activity at each step. Afrer the two chromatographic steps, the specific activity (U/~tg protein) of A1FI was increased 94-fold compared with the initial H. pylori sonic,ate. The fraction from third chromatography (size exclusion) which contained the peak AWl activity was visualized as one band on 12% SDS-pulyacrylaunde gel dectrophoresis. This indicated that AWl had been purified to homogeneity. The molecular weight of native and denatured AIFI were calculated by Superosechromatographyand SDS-PAGE. We conclude that 1) there is an protein produced by H. priori which can inlaibit acid secretion from gastric glands; 2) AIFI was purified to homogeneity; 3) AIF1 is a dimer with a 46 kDa subunit.
Immunology, Microbiology, and Inflammatory Disorders A839
• INCREASED ICAM-1 EXPRESSION ON HUMAN COLON EPITHELIAL CELLS IN RESPONSE TO BACTERIAL INVASION. G.T-J, |-inang, L. Eckmann, J. Fierer, M.F. Kagnoff. Dept. of Medicine, University of California, San Diego, La lolla, CA Human colon epithelial cells respond to invasive pathogens, including Salmonella dublin and Yersinia enterocolitica, with increased secretion of the cytokines IL-8, MCP-1, GM-CSF and TNFcc. These cytokines attract nentrophils and macrophages to the site of infection, and activate these cells locally. The adhesion molecule, ICAM-1, plays an important role in mediating adhesion of leukocytes to endothelial and other cells during their transit from the blood to the site of inflammation. ICAM-1 expression on endothelial and epithelial cells is known to be regulated by IFN-7 and TNFa, but little is known about other signals that are important in ICAM-1 regulation. Here we show increased ICAM-1 expression by epithelial cells in response to bacterial invasion. Methods: The human colon epithelial cell lines fir29 and T84 were co-cultured with the invasive bacteria S. dublin and Y. enterocolitica for 1 h, after which cells were incubated for 0.5-10 h in the presence of gentamicin to kill extracelhilar bacteria. Cells stained with FITC-labeled anti-human ICAM1 antibody were analyzed by flow eytometry. Results: ICAM-1 expression increased 4 h after infection of HT29 and T84 cells with S. dublin and Y. enterocolitica, and reached maximal levels 7-10 h after infection, with a 10-fold increase. No increase of ICAM-1 expression was observed after stimulation of cells with bacterial lipopolysaccharide. Conditioned media from S. dublin or Y. enterocolitica infected cells increased ICAM-I expression by HT29 cells, but not by T84 cells. This did not appear to be mediated by IFN*3, or TNFct since HT29 cells did not express IFN-T following bacterial invasion and increased ICAM-1 expression was not blocked by anti-TNF antibody. Conclusions: These data indicate that bacterial invasion of colon epithelial cells results in the upregulation of ICAM-1 expression. Increased levels of ICAM-1 on the cell surface may promote neutrophil adherence to bacteria-infected epithelial cells, and thereby aid in the destruction of bacteria that pass through those cells. Supported by NH-I grants DK35108 and DK47739, and a CCFA research fellowship.
IMMUNE MODULATION BY INTESTINAL EPITHELIAL CELL DIFFERENTIATION. N. Huang, D.R. Martin, and G.D. Wu. Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA Short chain fatty acids (SCFA) are normal products of anaerobic bacterial fermentation of carbohydrates in the colon and are the major source of nutrition for the human colonic epithelium. Butyrate, the SCFA most avidly metabolized by colonocytes, has recently been demonstrated to be an effective treatment for intestinal inflammatory diseases such ulcerative colitis. The mechanisms that lead to this response have not been well characterized. Intestinal inflammation leads to an alteration in patterns of epithelial differentiation with an increase in epithelial proliferation and an expansion of cell populations in an undifferentiated state. Recent studies by other investigators suggest that expansion of the undifferentiated crypt cell compartment may help to perpetuate the intestinal inflammatory response. Indeed, indirect evidence suggests that the state of epithelial cell differentiation may determine whether or not intestinal epithelia are capable of responding to an inflammatory stimulus. In order to elucidate how intestinal epithelial cells respond to inflammation, we have been studying the regulation of interleukin 8 (IL-8) gene expression in the Caco-2 intestinal cell line. IL-8 is a member of the human intercrine alpha subfamily of cytokines and is a selective chemoattractant and activator of neutrophils. We demonstrate that undifferentiated (pre-confluent) Caco-2 cells can be stimulated by 1L-113 to produce IL-8 mRNA. In contrast, however, differentiated (post-confluent) cells produce very little IL-8 mRNA after stimulation. Furthermore, induction of Caco-2 cell differentiation using the SCFAs butyrate and propionate led to a dose dependent inhibition of IL-8 mRNA expression. Preliminary studies suggest that the induction of histone hyperacetylation by these agents may play an operative role in this process. MCP-1, a member of a different class of chemoattractive cytokines, is also inhibited by Caco-2 cell differentiation at the level of mRNA abundance. Furthermore, we show that other classes of compounds that induce Caco-2 cell differentiation, such as vitamin D, also inhibit IL-8 mRNA expression. These results suggest that the inhibition of cytokine gene expression by intestinal cell differentiation may be a generalized process. The study of IL-8 gene regulation using these models may help to elucidate the cellular and molecular mechanisms that inhibit cytokine gene expression during intestinal epithelial differentiation.
• VIDEOENDOSCOPIC OBSERVATION OF ET-I INDUCED MUCOSAL ISCHEMIA: EFFECT OF AN ETA RECEPTOR SELECTIVE ANTAGONIST (FR139317). M. Hosokawa*, M. Tominaga÷, H. Nakamura ÷, T. Taniguchi*, S. Ueda÷, H. Tsukada*, M. Okuma ÷, M. Sakai + Kyoto Police Hospital*, Internal Medicine I. Kyoto University, Kyoto 606-01 Japan" Endothelin (ET) has been originally identified as a potent vasoconstrictive peptide in cultured media of porcine aortic endothelial cells. Recently, we reported that ET-1 enhanced both CI- secretion and muscle tension in rat colon. In this study, we determined how ET-1 induced mucosal damage in rat distal colon by real time observation with new videoendoscopic system (TOSHIBA TRE-300D, Japan). Furthermore we measured mucosal blood flow with laser doppler flowmetry (LDF) during control and condition period for ET-1. Methods: Carefully we washed distal colon of ether-anesthetized male rats with warm water and inserted videoendoscope. We kept observing colonic mucosa while and after sprinkling of ET-1 from biopsy channel with teflon tube. Mucosal blood flow was measured with LDF technique during endoscopy with flow probe inserted down the biopsy channel of endoscope. Results: Submucosal arteriole and venule contracted immediately after ET-1 administration (80 nmol/kg) and mueosal surface turned pale by the ischemic events. Approximately 50 rain took for both vessels to return normal in their diameter and color, showing blood reperfusion to mucosa. At this time small bleeding spots also appeared, which seemed to be extravasation localized inside the mucosa. Both LDF response to ET-I and histological findings were consistent with the videoendoscopic findings. All ET-l-induced changes were inhibited by pre-sprinkling of FR139317 (800 nmol/kg). Conclusion: Our results show that the videoendoscopes have so high resolution that we can detect mucosal ischemia induced by ET-1. FRi39317 inhibits effectively the ET-l-induced mucosal ischemia. FR139317 may provide a useful new therapy for inflammatory bowel diseases because local high concentration of ET-I is reportedly associated with this type of diseases. The new videoendoscope system was certified to be of great use for basic research of gastrointestinal pathophysiology.
• PURIFICATION AND CHARACTERIZATION OF AN ACIDINHIBITORY PROTEIN FROM HELICOBACTER PYLORI. L.L; Huang, D.R. Cave, and A.V. Kane. St. Elizabeth's Medical Center, and New England Medical Center, Tufts University Medical School, Boston, MA. Previous experiments have shown there are two factors produced by Helicobacter pylori that inhibit acid secretion from isolated gastric glands obtained from several mammaliaa species. Acid inhibitory factor 1 (AWl) is ddstroyed by pronase and heat. AIF2 is heat and pronase resistant and is of MW < 2kDa. This study describes the purification and characterization of AIF1 fromH, pylori. Six H. pylon isolates were cultured and tested for the production of AWl using the inhibition of uptake of 14C-amiunpyrine by secretogogue stimulated isolated rabbit gastric glands. The activity of AWl was defined as one unit of AWl being the amount required to inhibit the uptake of 14Caminopyrine by 50%. All isolates examined had inhibitory activity, in which the activity of AWl ranged from 1-65%. The most inhibitory isolate was then cultured under microaerophilic conditions in a fermenter, using brucella broth supplemented with 0.2% cyelodextrin to provide 10 L batches. The bacterial pellet was sonicated, and the supematant was then subjected to filtration through an Amicon membrane (MW cut off 10 kDa). The activity of AIFl remained in the retentate and AIF2 was in the filtrate. The retentate was then sequmtially subjected to hydroxyapatite, hydrephobic and size exclusion chromatography. Fractionswere tested for acid inhibitory activity at each step. Afrer the two chromatographic steps, the specific activity (U/~tg protein) of A1FI was increased 94-fold compared with the initial H. pylori sonic,ate. The fraction from third chromatography (size exclusion) which contained the peak AWl activity was visualized as one band on 12% SDS-pulyacrylaunde gel dectrophoresis. This indicated that AWl had been purified to homogeneity. The molecular weight of native and denatured AIFI were calculated by Superosechromatographyand SDS-PAGE. We conclude that 1) there is an protein produced by H. priori which can inlaibit acid secretion from gastric glands; 2) AIFI was purified to homogeneity; 3) AIF1 is a dimer with a 46 kDa subunit.
Immunology, Microbiology, and Inflammatory Disorders A839
• INCREASED ICAM-1 EXPRESSION ON HUMAN COLON EPITHELIAL CELLS IN RESPONSE TO BACTERIAL INVASION. G.T-J, |-inang, L. Eckmann, J. Fierer, M.F. Kagnoff. Dept. of Medicine, University of California, San Diego, La lolla, CA Human colon epithelial cells respond to invasive pathogens, including Salmonella dublin and Yersinia enterocolitica, with increased secretion of the cytokines IL-8, MCP-1, GM-CSF and TNFcc. These cytokines attract nentrophils and macrophages to the site of infection, and activate these cells locally. The adhesion molecule, ICAM-1, plays an important role in mediating adhesion of leukocytes to endothelial and other cells during their transit from the blood to the site of inflammation. ICAM-1 expression on endothelial and epithelial cells is known to be regulated by IFN-7 and TNFa, but little is known about other signals that are important in ICAM-1 regulation. Here we show increased ICAM-1 expression by epithelial cells in response to bacterial invasion. Methods: The human colon epithelial cell lines fir29 and T84 were co-cultured with the invasive bacteria S. dublin and Y. enterocolitica for 1 h, after which cells were incubated for 0.5-10 h in the presence of gentamicin to kill extracelhilar bacteria. Cells stained with FITC-labeled anti-human ICAM1 antibody were analyzed by flow eytometry. Results: ICAM-1 expression increased 4 h after infection of HT29 and T84 cells with S. dublin and Y. enterocolitica, and reached maximal levels 7-10 h after infection, with a 10-fold increase. No increase of ICAM-1 expression was observed after stimulation of cells with bacterial lipopolysaccharide. Conditioned media from S. dublin or Y. enterocolitica infected cells increased ICAM-I expression by HT29 cells, but not by T84 cells. This did not appear to be mediated by IFN*3, or TNFct since HT29 cells did not express IFN-T following bacterial invasion and increased ICAM-1 expression was not blocked by anti-TNF antibody. Conclusions: These data indicate that bacterial invasion of colon epithelial cells results in the upregulation of ICAM-1 expression. Increased levels of ICAM-1 on the cell surface may promote neutrophil adherence to bacteria-infected epithelial cells, and thereby aid in the destruction of bacteria that pass through those cells. Supported by NH-I grants DK35108 and DK47739, and a CCFA research fellowship.
IMMUNE MODULATION BY INTESTINAL EPITHELIAL CELL DIFFERENTIATION. N. Huang, D.R. Martin, and G.D. Wu. Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA Short chain fatty acids (SCFA) are normal products of anaerobic bacterial fermentation of carbohydrates in the colon and are the major source of nutrition for the human colonic epithelium. Butyrate, the SCFA most avidly metabolized by colonocytes, has recently been demonstrated to be an effective treatment for intestinal inflammatory diseases such ulcerative colitis. The mechanisms that lead to this response have not been well characterized. Intestinal inflammation leads to an alteration in patterns of epithelial differentiation with an increase in epithelial proliferation and an expansion of cell populations in an undifferentiated state. Recent studies by other investigators suggest that expansion of the undifferentiated crypt cell compartment may help to perpetuate the intestinal inflammatory response. Indeed, indirect evidence suggests that the state of epithelial cell differentiation may determine whether or not intestinal epithelia are capable of responding to an inflammatory stimulus. In order to elucidate how intestinal epithelial cells respond to inflammation, we have been studying the regulation of interleukin 8 (IL-8) gene expression in the Caco-2 intestinal cell line. IL-8 is a member of the human intercrine alpha subfamily of cytokines and is a selective chemoattractant and activator of neutrophils. We demonstrate that undifferentiated (pre-confluent) Caco-2 cells can be stimulated by 1L-113 to produce IL-8 mRNA. In contrast, however, differentiated (post-confluent) cells produce very little IL-8 mRNA after stimulation. Furthermore, induction of Caco-2 cell differentiation using the SCFAs butyrate and propionate led to a dose dependent inhibition of IL-8 mRNA expression. Preliminary studies suggest that the induction of histone hyperacetylation by these agents may play an operative role in this process. MCP-1, a member of a different class of chemoattractive cytokines, is also inhibited by Caco-2 cell differentiation at the level of mRNA abundance. Furthermore, we show that other classes of compounds that induce Caco-2 cell differentiation, such as vitamin D, also inhibit IL-8 mRNA expression. These results suggest that the inhibition of cytokine gene expression by intestinal cell differentiation may be a generalized process. The study of IL-8 gene regulation using these models may help to elucidate the cellular and molecular mechanisms that inhibit cytokine gene expression during intestinal epithelial differentiation.