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Superoxide production in DLD-1 and HT-29 cell lines and in primary epithelial cells from normal human colon
A98 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
664
666
MAXIMAL ACTIVATION OF EPITHELIAL NFKB - AN ADDITIONAL MOLECULAR MECHANISM UNDERLYING SYNERGY BETWEEN IFN-f AND TNF-A. Kenneth Nally, Joe O'Connell, John Morgan, Gerald C. O'Sullivan, John K. Collins, Fergus Lj Shanahan, Nui, Cork, Ireland. Background & Aim: The pleiotropic cytokines TNF-aand IFNyare associated with an excessive THI inflammatory infiltrate and immune dysregulation in Crohn' s disease. Previous reports have shown that they function synergistically to co-regulate gene expression in cells at receptor and post-receptor levels. These include the independent activation of NFKB by TNF-aand of IFN·responsive factors by IFNs with ensuing separate activation of genes related to the immunoinflammatory response. To further investigate possible molecular mechanisms underlying this synergism we examined the effects of IFNyon TNF-a-induced NFKB activation and pro-inflammatory gene regulation in human colonic HT29 epithelial cells. Methods: HT29 cells were treated with IFN-y(25ng/mL), TNF-a(5Ong/mL) or co-treated with IFNy+TNF-afor 30min, 2hr, 4hr, 8hr and 12hr. Activation of NFKB was assessed by immunofluorescence microscopy and mRNA levels for IKBa, the NFKB inhibitory molecule, were assessed by RT-PCR. Induction of the following NFKB regulated genes: cMyc, IKBa, PKR, COX2, ICAMI, TNFa, IL-8. IL-6 and HLA-DR were monitored at the mRNA and protein levels by semi-quantitative RT-PCR and by flow cytometry. Results: Co-treatment of HT29 with IFNy+TNFaresulted in significant nuclear staining for NFKB at 4hr, 8hr and 12hr. In contrast, neither IFN ynor TNF-aalone, elicited any detectable nuclear staining indicative of activation. In addition, IfNvtreatment alone did not significantly induce IL-8, TNF-a, COX2 or IKBaas detected by RT-PCR, whereas TNF-atreatment did induce all of the above genes as detected by RT-PCR and flow cytometry. However, co-treatment with both cytokines resulted in sustained and enhanced induction of all of the above genes, most notably in the case of ICAMI and cMyc, where there was a three fold log induction of the proteins as detected by flow cytometry. Conclusions: Functional synergy between IFNyand TNF-aoccurs at several levels. The present results provide further evidence for co-operation between the two cytokines within colonic epithelial cells at the level of gene expression with maximal and sustained induction of inflammatory gene expression and synergistic induction of the NFKB pathway.
EXPRESSION OF CD14 ON HUMAN INTESTINAL EPITHELIAL CELL LINES. David Petra Funda, Ludmila Tuckova, Maria Antoinetta Farre, T. Iwase, Itaru Moro, Helena Tlaskalova, Inst Microbial, Czech Acad of Sci, Dept Immunol, Prague, CZ, Czech Republic; Inst Microbial, Acad of Sci, Dept Immunol, Prague, CZ, Czech Republic; Dept of Pathology, Nihon Univ Sch of Dentistry, Tokyo, Japan.
665 SUPEROXIDE PRODUCTION IN DLD-l AND HT-29 CELL LINES AND IN PRIMARY EPITHELIAL CELLS FROM NORMAL HUMAN COLON. Anders Perner, Lars Andresen, Gitte Pedersen, Torben Saermark, Jorn Brynskov, Jorgen Rask-Madsen, Dept of Gastroenterology, Herlev Univ Hosp, Copenhagen, Denmark. Background: Generation of the superoxide radical (0;) by mucosal inflammatory cells has been suggested to play a pathophysiological role in chronic inflammatory bowel disease. An epithelial source of 0; was suggested by the description of nitrotyrosine, a marker of 0; and nitric oxide co-production, in the inflamed epithelium.l" 0; is synthesized by NADPH oxidase both in normal and diseased states in a variety of cell types, but has never been demonstrated in colonic epithelial cells. Aims: To measure 0; production, if any, in colonic epithelial cell lines and in epithelial cells from the normal human colon. Methods: DLD-I and HT-29 cells were grown to confluence in 24-well plates in Dulbecco's modified Eagle medium (DMEM). In ten subjects with uninflamed bowel, seven biopsy specimens were taken during colonoscopy. The epithelium was isolated by EDTNEGTA treatment for 10 minutes and incubated in DMEM for I hour. Basal and NADPH (lmM)-induced production of 0; was measured by superoxide dismutase (SOD)-inhibitable (200 U/mL) reduction of cytochrome c (0.5 mg/mL) or by SOD-inhibitable luminescence of luminol (500 /LM) and adjusted to protein contents. Results: Basal production of 0; was undetectable in the cell lines, in contrast to the primary cells, see table. NADPH induced 0; production in the cell lines and in the primary cells a three-fold increase was observed. Using luminescence technique, this was observed within seconds although cell membranes are impermeable to NADPH. Conclusions: These results show that human colonic epithelial cells produce 0;, possibly through an NADPH oxidase localized to the outer cell membrane. 1. Gastroenterology 1996; 11:871. 2. Gut 1999;45:AI24
Basal and NADPH-induced Superoxide Production inHuman Colonic Epithelial Cells DLD-1 Cells Cytochrome c reduction Basal, nmol/h/mg, n=5 NADPH, nmol/h/mg, n=5 Luminescence Basal, RLUlmg, n=5 NADPH, RLU/mg, n=5
HT29 Cells
Background/Aim: Some intestinal epithelial cell lines as well as endothelial cell were shown to respond to LPS. However, the expression ofCDI4 on these cells and the release of soluble CD 14 by these cells has not been studied in detail. Aim of this study was to investigate the expression of LPS receptors and responsiveness to endotoxin of three human adenocarcinoma cell lines SW-480, HT-29 and Caco-2. Methods: Immunofluorescence (FACS), Western blot and RT-PCR were used to detect expression of CDI4 on cell membrane, protein and messenger RNA level of cultivated intestinal epithelial cell lines. Results: FACS analysis revealed no expression of CDllc CDI8 but low expression of CDI4 on HT-29, and Caco-2 epithelial cell lines (ECLs), whereas the expression of CDI4 on SW-480 was questionable. However, Western blot analysis of supernatans from phosphatidylinositol-specific phospholipase C treated cells displayed a clear band of about 55 kDa from both HT-29 and Caco-2 as a well as SW-480 ECLs. Using two different primer combinations CDI4 mRNA was found in HT-29 cell line irrespective of exposure to LPS 0.01, 0.1, I, 10 /Lg ml. Messenger RNA for CDI4 was detected also in SW-480 and Caco-2 ECLs. Interestingly, CDI4 was found in tissue culture supernatants of all three cell lines. Finally, only HT-29 and SW-480 but not Caco-2 responded to exposure to OJ, 1, 10 and 100 /Lg/ml of LPS by IL8 release. Conclusion: We found that HT-29, Caco-2 and SW-480 human intestinal ECLs do express low levels of mCDI4. The fact that intestinal ECLs are capable of expression and release of CDI4 could have important implications in modulating the interaction of intestinal mucosa with bacterial flora present in the gut.
667 RELATION BETWEEN ADHERENCE OF LACTOBACILLUS TO COLONIC EPITHELIUM AND ITS ABILITY TO COLONIZE THE GUT. Maria C. Antolin, Ana M. Garcia-Lafuente, Francisco Guamer, Ernesto Crespo, Antonia Andreu, Jordi Osset, Juan Ramon Malagelada, Digest System Research Unit Hosp Gen Vall d'Hebron, Barcelona, Spain; Microbiological Service Hosp Gen Vall d'Hebron, Barcelona, Spain. The ability of certain bacterial strains to adhere to mucosal surfaces could confere them an important competitive advantage over other strains for the colonization of the gut. However, the precise role of bacterial adherence in this process is not well established. Our aim was to investigate putative relations between adherence of different strains of Lactobacillus to colonic epithelium and Lactobacillus survival in the colon. Seven strains of Lactobacillus were studied: 4 strains of L. crispatus, two of them from human vaginal flora iLcrl y Lcr2) and two from intestinal flora (Lcd y Lcr4), I strain of L. jensenii from human vaginal flora (Lj), I L. acidophilus from intestinal flora (La) and I strain of L. delbrueckii subsp bulgaricus (£d). In the adhesion assay 2 x 105 freshly isolated rat colonocytes were incubated with 107 bacteria of a single strain, at 3rC in a shaking bath. After 30 minutes, cells were washed to eliminate non-adhering bacteria and Gram staining was performed. Bacterial adherence (Adh) was quantified as the number of attached bacteria per cell after counting 50 cells in each of 4 separate trials. To study the ability of the different Lactobacillus to colonize the intestinal tract we used our previously described model of colonic segment excluded from the fecal transit in the rat (Am J Physiol 1997; 272(35):GIO-GI5). After elimination of the native flora by antibiotic washings, the segment was colonized with a single bacterial strain. We used seven groups of animals, and each group was colonized with one of the seven strains of Lactobacillus. Forty-eight hours after instillation of each strain, animals were euthanized and colonization was assessed by microbiological culture of the homogenized tissue. The percentage of animals with positive colonization (Col) was calculated in each group. The results obtained are summarized in the table. Conclusion: Adherence of Lactobacillus to colonic epithelial cells is a determinant factor of their ability to colonize the intestinal tract in vivo.
PrlmaryCelis
UD 5± l'
UD 4± r-
39± 22 123±16'
UD UD
UD UD
9099 ± 2365 24518 ± 7410'
Values are mean (± SEM) production rates adjusted tomg protein. RLU, relative light units; UD, undetectable. 'P<0.05 compared with basal (paired t-test).
Col
Adh
LJ
Lcr2
Lcr1
La
Lcr3
Lcr4
Ld
100% 6.3±.52
100% 4.4±.71
100% 3.6±.42
80% 2.5±.33
40% 2.4±55
18% 1.9±.29
0% 0.4±.07
Bacterial adherence (Adh) isexpressed as mean ± SEM
GASTROENTEROLOGY Vol. 118, No.4
664
666
MAXIMAL ACTIVATION OF EPITHELIAL NFKB - AN ADDITIONAL MOLECULAR MECHANISM UNDERLYING SYNERGY BETWEEN IFN-f AND TNF-A. Kenneth Nally, Joe O'Connell, John Morgan, Gerald C. O'Sullivan, John K. Collins, Fergus Lj Shanahan, Nui, Cork, Ireland. Background & Aim: The pleiotropic cytokines TNF-aand IFNyare associated with an excessive THI inflammatory infiltrate and immune dysregulation in Crohn' s disease. Previous reports have shown that they function synergistically to co-regulate gene expression in cells at receptor and post-receptor levels. These include the independent activation of NFKB by TNF-aand of IFN·responsive factors by IFNs with ensuing separate activation of genes related to the immunoinflammatory response. To further investigate possible molecular mechanisms underlying this synergism we examined the effects of IFNyon TNF-a-induced NFKB activation and pro-inflammatory gene regulation in human colonic HT29 epithelial cells. Methods: HT29 cells were treated with IFN-y(25ng/mL), TNF-a(5Ong/mL) or co-treated with IFNy+TNF-afor 30min, 2hr, 4hr, 8hr and 12hr. Activation of NFKB was assessed by immunofluorescence microscopy and mRNA levels for IKBa, the NFKB inhibitory molecule, were assessed by RT-PCR. Induction of the following NFKB regulated genes: cMyc, IKBa, PKR, COX2, ICAMI, TNFa, IL-8. IL-6 and HLA-DR were monitored at the mRNA and protein levels by semi-quantitative RT-PCR and by flow cytometry. Results: Co-treatment of HT29 with IFNy+TNFaresulted in significant nuclear staining for NFKB at 4hr, 8hr and 12hr. In contrast, neither IFN ynor TNF-aalone, elicited any detectable nuclear staining indicative of activation. In addition, IfNvtreatment alone did not significantly induce IL-8, TNF-a, COX2 or IKBaas detected by RT-PCR, whereas TNF-atreatment did induce all of the above genes as detected by RT-PCR and flow cytometry. However, co-treatment with both cytokines resulted in sustained and enhanced induction of all of the above genes, most notably in the case of ICAMI and cMyc, where there was a three fold log induction of the proteins as detected by flow cytometry. Conclusions: Functional synergy between IFNyand TNF-aoccurs at several levels. The present results provide further evidence for co-operation between the two cytokines within colonic epithelial cells at the level of gene expression with maximal and sustained induction of inflammatory gene expression and synergistic induction of the NFKB pathway.
EXPRESSION OF CD14 ON HUMAN INTESTINAL EPITHELIAL CELL LINES. David Petra Funda, Ludmila Tuckova, Maria Antoinetta Farre, T. Iwase, Itaru Moro, Helena Tlaskalova, Inst Microbial, Czech Acad of Sci, Dept Immunol, Prague, CZ, Czech Republic; Inst Microbial, Acad of Sci, Dept Immunol, Prague, CZ, Czech Republic; Dept of Pathology, Nihon Univ Sch of Dentistry, Tokyo, Japan.
665 SUPEROXIDE PRODUCTION IN DLD-l AND HT-29 CELL LINES AND IN PRIMARY EPITHELIAL CELLS FROM NORMAL HUMAN COLON. Anders Perner, Lars Andresen, Gitte Pedersen, Torben Saermark, Jorn Brynskov, Jorgen Rask-Madsen, Dept of Gastroenterology, Herlev Univ Hosp, Copenhagen, Denmark. Background: Generation of the superoxide radical (0;) by mucosal inflammatory cells has been suggested to play a pathophysiological role in chronic inflammatory bowel disease. An epithelial source of 0; was suggested by the description of nitrotyrosine, a marker of 0; and nitric oxide co-production, in the inflamed epithelium.l" 0; is synthesized by NADPH oxidase both in normal and diseased states in a variety of cell types, but has never been demonstrated in colonic epithelial cells. Aims: To measure 0; production, if any, in colonic epithelial cell lines and in epithelial cells from the normal human colon. Methods: DLD-I and HT-29 cells were grown to confluence in 24-well plates in Dulbecco's modified Eagle medium (DMEM). In ten subjects with uninflamed bowel, seven biopsy specimens were taken during colonoscopy. The epithelium was isolated by EDTNEGTA treatment for 10 minutes and incubated in DMEM for I hour. Basal and NADPH (lmM)-induced production of 0; was measured by superoxide dismutase (SOD)-inhibitable (200 U/mL) reduction of cytochrome c (0.5 mg/mL) or by SOD-inhibitable luminescence of luminol (500 /LM) and adjusted to protein contents. Results: Basal production of 0; was undetectable in the cell lines, in contrast to the primary cells, see table. NADPH induced 0; production in the cell lines and in the primary cells a three-fold increase was observed. Using luminescence technique, this was observed within seconds although cell membranes are impermeable to NADPH. Conclusions: These results show that human colonic epithelial cells produce 0;, possibly through an NADPH oxidase localized to the outer cell membrane. 1. Gastroenterology 1996; 11:871. 2. Gut 1999;45:AI24
Basal and NADPH-induced Superoxide Production inHuman Colonic Epithelial Cells DLD-1 Cells Cytochrome c reduction Basal, nmol/h/mg, n=5 NADPH, nmol/h/mg, n=5 Luminescence Basal, RLUlmg, n=5 NADPH, RLU/mg, n=5
HT29 Cells
Background/Aim: Some intestinal epithelial cell lines as well as endothelial cell were shown to respond to LPS. However, the expression ofCDI4 on these cells and the release of soluble CD 14 by these cells has not been studied in detail. Aim of this study was to investigate the expression of LPS receptors and responsiveness to endotoxin of three human adenocarcinoma cell lines SW-480, HT-29 and Caco-2. Methods: Immunofluorescence (FACS), Western blot and RT-PCR were used to detect expression of CDI4 on cell membrane, protein and messenger RNA level of cultivated intestinal epithelial cell lines. Results: FACS analysis revealed no expression of CDllc CDI8 but low expression of CDI4 on HT-29, and Caco-2 epithelial cell lines (ECLs), whereas the expression of CDI4 on SW-480 was questionable. However, Western blot analysis of supernatans from phosphatidylinositol-specific phospholipase C treated cells displayed a clear band of about 55 kDa from both HT-29 and Caco-2 as a well as SW-480 ECLs. Using two different primer combinations CDI4 mRNA was found in HT-29 cell line irrespective of exposure to LPS 0.01, 0.1, I, 10 /Lg ml. Messenger RNA for CDI4 was detected also in SW-480 and Caco-2 ECLs. Interestingly, CDI4 was found in tissue culture supernatants of all three cell lines. Finally, only HT-29 and SW-480 but not Caco-2 responded to exposure to OJ, 1, 10 and 100 /Lg/ml of LPS by IL8 release. Conclusion: We found that HT-29, Caco-2 and SW-480 human intestinal ECLs do express low levels of mCDI4. The fact that intestinal ECLs are capable of expression and release of CDI4 could have important implications in modulating the interaction of intestinal mucosa with bacterial flora present in the gut.
667 RELATION BETWEEN ADHERENCE OF LACTOBACILLUS TO COLONIC EPITHELIUM AND ITS ABILITY TO COLONIZE THE GUT. Maria C. Antolin, Ana M. Garcia-Lafuente, Francisco Guamer, Ernesto Crespo, Antonia Andreu, Jordi Osset, Juan Ramon Malagelada, Digest System Research Unit Hosp Gen Vall d'Hebron, Barcelona, Spain; Microbiological Service Hosp Gen Vall d'Hebron, Barcelona, Spain. The ability of certain bacterial strains to adhere to mucosal surfaces could confere them an important competitive advantage over other strains for the colonization of the gut. However, the precise role of bacterial adherence in this process is not well established. Our aim was to investigate putative relations between adherence of different strains of Lactobacillus to colonic epithelium and Lactobacillus survival in the colon. Seven strains of Lactobacillus were studied: 4 strains of L. crispatus, two of them from human vaginal flora iLcrl y Lcr2) and two from intestinal flora (Lcd y Lcr4), I strain of L. jensenii from human vaginal flora (Lj), I L. acidophilus from intestinal flora (La) and I strain of L. delbrueckii subsp bulgaricus (£d). In the adhesion assay 2 x 105 freshly isolated rat colonocytes were incubated with 107 bacteria of a single strain, at 3rC in a shaking bath. After 30 minutes, cells were washed to eliminate non-adhering bacteria and Gram staining was performed. Bacterial adherence (Adh) was quantified as the number of attached bacteria per cell after counting 50 cells in each of 4 separate trials. To study the ability of the different Lactobacillus to colonize the intestinal tract we used our previously described model of colonic segment excluded from the fecal transit in the rat (Am J Physiol 1997; 272(35):GIO-GI5). After elimination of the native flora by antibiotic washings, the segment was colonized with a single bacterial strain. We used seven groups of animals, and each group was colonized with one of the seven strains of Lactobacillus. Forty-eight hours after instillation of each strain, animals were euthanized and colonization was assessed by microbiological culture of the homogenized tissue. The percentage of animals with positive colonization (Col) was calculated in each group. The results obtained are summarized in the table. Conclusion: Adherence of Lactobacillus to colonic epithelial cells is a determinant factor of their ability to colonize the intestinal tract in vivo.
PrlmaryCelis
UD 5± l'
UD 4± r-
39± 22 123±16'
UD UD
UD UD
9099 ± 2365 24518 ± 7410'
Values are mean (± SEM) production rates adjusted tomg protein. RLU, relative light units; UD, undetectable. 'P<0.05 compared with basal (paired t-test).
Col
Adh
LJ
Lcr2
Lcr1
La
Lcr3
Lcr4
Ld
100% 6.3±.52
100% 4.4±.71
100% 3.6±.42
80% 2.5±.33
40% 2.4±55
18% 1.9±.29
0% 0.4±.07
Bacterial adherence (Adh) isexpressed as mean ± SEM