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Sequential expression of transforming growth factor β1 (TGFβ1), transforming growth factor β3 (TGFβ3) and transforming growth factor alpha (TGFα) in patients with Crohn's disease (CD)
April 1998 inflammatory cells. In the present study, we report on several cytokines secreted in response to stimulation with lipopoysaccharides (LPS) and TGF[31, as well as lymphocyte restricted cytokines. Methods: Constitutive secretion of cytokines (IL-113, IL-6, IL-7, IL-8, IL-10, and GM-CSF) by 6 pancreatic epithelial duct cell lines (AsPC-1, BxPC-3, Capan-1, Capan-2, PANC-1, and PaCa 44) was investigated by the ELISA technique. Furthermore, we stimulated these 6 cell lines with different concentrations of LPS, TGFI31, IL-4 and IFNy. After 48 hours, the concentrations of IL-113, IL-6, IL-8, and GM-CSF were determined in supematants of cell cultures. Results: All cell lines produced TGFI31 and IL-8. No production of IL-7 and IL-10 could be found. IL-113 was secreted only by BxPC-3. Capan-1, BxPC-3, and PaCa 44 secreted IL-6. GM-CSF was secreted by Capan-1, Capan-2 and PaCa 44. After stimulation with LPS and TGFI31 the secretion of all investigated cytokines, was elevated. IL-4 stimulated secretion Of IL-8 and IFN'/ induced IL-6 secretion in a dose dependent manner. Conclusion: Pancreatic duct epithelial cells secreted TGFI31, IL-1, IL-6, IL-8, and GM-CSF. Cytokine secretion could be regulated by LPS and TGFI3. There was a selective stimulation of different cytokines using IL-4 as well as IFN'/. These results suggest epithelial cells play an important role during pancreas inflammation by influencing the cytokine network. Supported by BMBF, FKZ 01/ZZ/9102 and NIH, DK 28537 • G3987 PROINFLAMMATORY CYTOKINES DIFFERENTIALLY AFIYECT MUCIN EXPRESSION IN LS180 CELLS. M.-L Enss, R. Eisenbl~itter, M. Henrichs, S. Wagner, M. Comberg, W. Bell, H.J. Hedrich. Dept. Lab. Anita. Sci.; Dept. Gastroenterol. Hepatol.; Dept. Pharmacol.; Medical School. Hannover, D-30623, Germany.
Inflammatory cytokines play a key role in the pathogenesis of inflammatory bowel diseases, however, their effects on protective mucins are not fully understood. This study describes the mucin expression of a human colon cancer cell line in response to proinflammatory cytokines on MUC-mRNA and glycoprotein levels. Methods: LS180 cells (ATCC) were incubated with IL-1, IL-6, and TNFc¢ (1 to 10 ng/ml) for 1 to 16 h in RPMI 1640 medium. Expression of MUC2, MUCSAC, MUCSB and MUC6 mRNA was analysed by semiquantitatlve RT-PCR, using GAPDH a s a housekeeping gene. Mucins discharged in response to cytokines were isolated by gel filtration (Sepharose CL-4B), and their carbohydrate and protein contents were quantitated by PAS and Bradford reaction, respectively. Cytokine effects were calculated with respect to the particular cell density in % of controls. Results: All proinflammatory cytokines down-regulated the mRNA expression of MUC2 and up-regulated the mRNA expression of MUCSAC, MUCSB and MUC6 in a tlme-dependeet manner. TNFct was significantly more effective than IL-1 and IL-6. Maximal mRNA expression preceded the peak of mucin release by 4 to 8 hours. While IL-1 and IL-6 predominantly upregulated the release of mucin proteins (+43% and +33% after 8 h incubation, respectively), TNF~ (+69% after 8 h), in addition, stimulated the glycosylation of mucin carbohydrates (+53% after 8 h). Conclusion: Proinflammatory cytokines regulate mucin secretion in a colon cell line by differential induction of MUC-gene expression. Mucins initiated by one of the cytokines display a differing degree of glycosylation and thereby may imply functional modifications. Supported by the Deutsche Forschungsgemeinschafi, SFB 280 • G3988 SEQUENTIAL EXPRESSION OF TRANSFORMING GROWTH FACTOR [31 (TGFI31), TRANSFORMING GROWTH FACTOR 133 (TGF[33) AND TRANSFORMING GROWTH FACTOR ALPHA (TGF~) IN PATIENTS WITH CROHN'S DISEASE (CD). H. Ernst, U. Hoffmann*, J. Eisenach, S. Wedel, M. Dietl*, S. Schreiber, H. Lochs and the IL-10 study group. Depts. of Medicine IV and Pathology , , CharitY, Berlin, Germany. Background: The transforming growth factor family plays a key role in wound repair. Differential expression of transforming growth factors in colonic mucosa could affect healing and the course of CD. Here we compared the expression of TGF]31, TGF[33, and TGF~ in colonic epithelial ceils in endoscopically affected and normal appearing areas in Pat. with active CD before and after 4 weeks of treatment with IL-10. Method: 11 pts. with chronic active steroid dependent CD (5 male, 6 female) were colonoscoped and routine biopsies were taken from affected and normal appearing areas. Pts. were treated at the Charit6 Univ. Hospital in addition to concurrent treatmeut with steroids with IL-10 at different doses (4-20ug/kg bodyweight s.c. for 4 weeks). Paraffin serial sections were stained with antibodies to a proliferation marker (KI-67, Oncogene Science), TGF[H (R&D Systems), TGF[33 (Oncogene Science) and TGFc~ (Oncogene Science) using the ABC method. Coded specimens were assessed for the number of positive epithelial cells for Ki-67 (300 consecutive cells in the proliferation zone). Staining intensitiy (0-3) for TGF[31, TGFI33 and TGFc~ were assessed in 300 consecutive epithelial cells. Results: Expression of proliferation marker and TGF[31 was increased in inflamed mucosa (mean staining intensitiy ± SEM; 136 _+37) compared to noninflamed mucosa (TGF[31, 103 ± 40) before IL-10 treatment but was not changed after four weeks of IL-10 treatment. Expression of TGFc~ in intact mucosa was decreased after 4 weeks treatment with IL-10 (after 4 weeks: inflamed mucosa; 158+28 vs 125+35 non inflamed mucosa). Expression of TGF[33 was significantly decreased in intact mucosa after 4 weeks of treatment with IL-10 (affected mucosa; 51+17 vs 12+4 in intact muc0sa, p<0.05). Conclusion: Regeneration is increased in active CD.
Immunology, Microbiology, and Inflammatory Disorders A973 TGFI~I and TGFct are increased in inflamed mucosa and expression of TGFI31 is not changed after a treatment of 4 weeks with IL-10. G3989 ARE PROTEIN FERMENTATION METABOLITES INVOLVED IN TIlE PAHOGENESIS OF ULCERATIVE COLITIS? P. Evenepoel, D. Claus, B. Geypens, Y. Ghoos and P. Rutgeerts. Center for Gastrointestinal Research, University of Leuven, B-3000 Belgium.
Introduction: Considerable evidence suggests that hydrogen sulphide (H2S) and phenols are involved in the pathogenesis of ulcerative colitis (UC). It is hypothesised that these metabolites initiate and perpetuate epithelial injury and/or dysfunction, thereby leading to unrestrained mucosal inflammation in a susceptible host. Colonic H2S is derived both from the fermentation of unabsorbed sulfur amino acids and the reduction of (inorganic) sulfate. Phenols are unique endproducts of the fermentation of unabsorbed tyrosine. The major phenols in the colonic lumen are phenol and p-cresol. Most of the phenols are absorbed, detoxified and excreted in urine. Aim: The aim of the present study was to evaluate whether fecal concentration and/or urinary output of phenols are increased in UC patients. Methods: 27 healthy volunteers and 28 patients with UC were studied. All patients were treated either with steroids, 5-ASA, azathioprine, cyclosporine or a combination of these. Disease activity in the patients, as assessed using the colitis activity index ranged from 0 to 14 (mean ± SEM: 5.1 ± 0.7). 24h-urine collections and fecal samples were analysed for phenol and p-cresol by liquid-liquid extraction and gaschromatography-mass spectrometry. Results: The results are summarised in the table. Both the fecal concentration and the urinary output of phenols were significantly decreased in UC patients. Neither the fecal concentration, nor the urinary output of phenols correlated with disease activity. Conclusion: It is unlikely that endproducts of protein fermentation play an initiating or perpetuating role in the disease process of UC. Feces Urine
(mg/kg wet weight) (mg/d dry weight) (mg/d)
Controls 112 ± 12 0.38 ± 0.05 51 ± 6
UC patients 14 ± 5 0.08 ± 0.02 34 ± 5
P < 0.0001 < 0.0001 < 0.05
G3990 IN VIVO FLUORESCENCE MICROSCOPY FOR ASSESSMENT OF LEUKOCYTE-ENDOTHELIAL-INTERACTION IN THE LEFT COLON IN DEXTRAN SULPHATE SODIUM [DSS]-INDUCED COLITIS. S.Farkas. H.Herfarth*, M Steinbaner, M. Anthuber. Department of Surgery, Regensburg, *Department of Internal Med I, Regensburg. Background: Leukocyte adhesion plays a pivotal role in acute and chronic inflammatory bowel disease. Therefore, the objective of this study was to determine leukocyte-endothelial-interaction in intramural collecting venules and submucosal postcapillary venules of the colon by means of in vivo microscopy in DSS induced colitis in mice. Methods Female Balb/c mice, weighing 20 ± 0,3g were used for experiments. Control mice received normal drinking water. Chronic colitis was established after four cycles of feeding 5% DSS -dissolved in their drinking water- for 7 days and normal water for 10 days. Five weeks after the last DSS cycle in vivo microscopy was performed according to the technique by Massberg et al (1) and Gonzalez et al (2) described for small bowel isografts. In anaesthetized mice, at a stable mean arterial pressure above 80 mm Hg, the left colon was mobilized and placed on a specially designed stage. Colonic microcirculatory networks were visualized by i.v. injection of FITC labeled Dextran (2,0pmol/kg). Rhodamin 6G (0,1tamol/kg) was injected, as fluorescent dye to stain leukocytes in vivo. Epi-illumination technique was used with 680 fold magnification. Permanent leukocyte adherence ("sticker") and temporary leukocyte adherence ("roller") were assessed in 10 randomly selected collecting venules and postcapillary venules. Leukocytes adhering longer than 30 sec to the endothelium were determined as stickers (n/mm2 venular surface). Rolling leukocytes were defined as cells moving along the vessel wall with a velocity of less than 30% of the central stream (% of rollers of all free - moving leukocytes during the observation period of 30 see). Results: In mice with chronic colitis we found a significant increase of leukocyte sticking and rolling in submucosal postcapillary (PV) and in intramural collecting venules (CV) compared to controls. Moreover we found an enhanced leukocyte accumulation in postcapillary venules than in collecting venules (p=0,005) in mice with DSS induced colitis.
albC n = 6 alb C + DSS n=6
I sticker in PV sticker in CV (n/mm2 venular surface) 8.3±3.5 2.9±1.8 171.4 ± 29.4*
55.6 ± 13.4"
roller in PV roller in CV (% of all moving leukocytes) 49.7±1.4 42.13±3.8 84.9 ± 3.2*
70.73 ± 4.9*
Mean _+SEM; * = p < 0.01 vs. Balb C Conclusion: We propose a model for assessment of leukocyte-endothelial interaction in the microcirculation of the colon in DSS induced chronic colitis. Leukocyte roiling and sticking in colonic postcapillary and collecting venules is considerably increased in DSS induced chronic colitis, reflecting the degree of inflammation. In addition leukocyte adhesion is most prominent in submucosal postcapillary vessels localized closest to inflammation site. This model may offer a novel approach for quantification of leukocyteendothelial-interaction in experimental inflammatory bowel disease. [1] Massberg et al. Surgery in press 2. Gonzalez et al. Transplantation 1994; (58) 403-8)
Inflammatory cytokines play a key role in the pathogenesis of inflammatory bowel diseases, however, their effects on protective mucins are not fully understood. This study describes the mucin expression of a human colon cancer cell line in response to proinflammatory cytokines on MUC-mRNA and glycoprotein levels. Methods: LS180 cells (ATCC) were incubated with IL-1, IL-6, and TNFc¢ (1 to 10 ng/ml) for 1 to 16 h in RPMI 1640 medium. Expression of MUC2, MUCSAC, MUCSB and MUC6 mRNA was analysed by semiquantitatlve RT-PCR, using GAPDH a s a housekeeping gene. Mucins discharged in response to cytokines were isolated by gel filtration (Sepharose CL-4B), and their carbohydrate and protein contents were quantitated by PAS and Bradford reaction, respectively. Cytokine effects were calculated with respect to the particular cell density in % of controls. Results: All proinflammatory cytokines down-regulated the mRNA expression of MUC2 and up-regulated the mRNA expression of MUCSAC, MUCSB and MUC6 in a tlme-dependeet manner. TNFct was significantly more effective than IL-1 and IL-6. Maximal mRNA expression preceded the peak of mucin release by 4 to 8 hours. While IL-1 and IL-6 predominantly upregulated the release of mucin proteins (+43% and +33% after 8 h incubation, respectively), TNF~ (+69% after 8 h), in addition, stimulated the glycosylation of mucin carbohydrates (+53% after 8 h). Conclusion: Proinflammatory cytokines regulate mucin secretion in a colon cell line by differential induction of MUC-gene expression. Mucins initiated by one of the cytokines display a differing degree of glycosylation and thereby may imply functional modifications. Supported by the Deutsche Forschungsgemeinschafi, SFB 280 • G3988 SEQUENTIAL EXPRESSION OF TRANSFORMING GROWTH FACTOR [31 (TGFI31), TRANSFORMING GROWTH FACTOR 133 (TGF[33) AND TRANSFORMING GROWTH FACTOR ALPHA (TGF~) IN PATIENTS WITH CROHN'S DISEASE (CD). H. Ernst, U. Hoffmann*, J. Eisenach, S. Wedel, M. Dietl*, S. Schreiber, H. Lochs and the IL-10 study group. Depts. of Medicine IV and Pathology , , CharitY, Berlin, Germany. Background: The transforming growth factor family plays a key role in wound repair. Differential expression of transforming growth factors in colonic mucosa could affect healing and the course of CD. Here we compared the expression of TGF]31, TGF[33, and TGF~ in colonic epithelial ceils in endoscopically affected and normal appearing areas in Pat. with active CD before and after 4 weeks of treatment with IL-10. Method: 11 pts. with chronic active steroid dependent CD (5 male, 6 female) were colonoscoped and routine biopsies were taken from affected and normal appearing areas. Pts. were treated at the Charit6 Univ. Hospital in addition to concurrent treatmeut with steroids with IL-10 at different doses (4-20ug/kg bodyweight s.c. for 4 weeks). Paraffin serial sections were stained with antibodies to a proliferation marker (KI-67, Oncogene Science), TGF[H (R&D Systems), TGF[33 (Oncogene Science) and TGFc~ (Oncogene Science) using the ABC method. Coded specimens were assessed for the number of positive epithelial cells for Ki-67 (300 consecutive cells in the proliferation zone). Staining intensitiy (0-3) for TGF[31, TGFI33 and TGFc~ were assessed in 300 consecutive epithelial cells. Results: Expression of proliferation marker and TGF[31 was increased in inflamed mucosa (mean staining intensitiy ± SEM; 136 _+37) compared to noninflamed mucosa (TGF[31, 103 ± 40) before IL-10 treatment but was not changed after four weeks of IL-10 treatment. Expression of TGFc~ in intact mucosa was decreased after 4 weeks treatment with IL-10 (after 4 weeks: inflamed mucosa; 158+28 vs 125+35 non inflamed mucosa). Expression of TGF[33 was significantly decreased in intact mucosa after 4 weeks of treatment with IL-10 (affected mucosa; 51+17 vs 12+4 in intact muc0sa, p<0.05). Conclusion: Regeneration is increased in active CD.
Immunology, Microbiology, and Inflammatory Disorders A973 TGFI~I and TGFct are increased in inflamed mucosa and expression of TGFI31 is not changed after a treatment of 4 weeks with IL-10. G3989 ARE PROTEIN FERMENTATION METABOLITES INVOLVED IN TIlE PAHOGENESIS OF ULCERATIVE COLITIS? P. Evenepoel, D. Claus, B. Geypens, Y. Ghoos and P. Rutgeerts. Center for Gastrointestinal Research, University of Leuven, B-3000 Belgium.
Introduction: Considerable evidence suggests that hydrogen sulphide (H2S) and phenols are involved in the pathogenesis of ulcerative colitis (UC). It is hypothesised that these metabolites initiate and perpetuate epithelial injury and/or dysfunction, thereby leading to unrestrained mucosal inflammation in a susceptible host. Colonic H2S is derived both from the fermentation of unabsorbed sulfur amino acids and the reduction of (inorganic) sulfate. Phenols are unique endproducts of the fermentation of unabsorbed tyrosine. The major phenols in the colonic lumen are phenol and p-cresol. Most of the phenols are absorbed, detoxified and excreted in urine. Aim: The aim of the present study was to evaluate whether fecal concentration and/or urinary output of phenols are increased in UC patients. Methods: 27 healthy volunteers and 28 patients with UC were studied. All patients were treated either with steroids, 5-ASA, azathioprine, cyclosporine or a combination of these. Disease activity in the patients, as assessed using the colitis activity index ranged from 0 to 14 (mean ± SEM: 5.1 ± 0.7). 24h-urine collections and fecal samples were analysed for phenol and p-cresol by liquid-liquid extraction and gaschromatography-mass spectrometry. Results: The results are summarised in the table. Both the fecal concentration and the urinary output of phenols were significantly decreased in UC patients. Neither the fecal concentration, nor the urinary output of phenols correlated with disease activity. Conclusion: It is unlikely that endproducts of protein fermentation play an initiating or perpetuating role in the disease process of UC. Feces Urine
(mg/kg wet weight) (mg/d dry weight) (mg/d)
Controls 112 ± 12 0.38 ± 0.05 51 ± 6
UC patients 14 ± 5 0.08 ± 0.02 34 ± 5
P < 0.0001 < 0.0001 < 0.05
G3990 IN VIVO FLUORESCENCE MICROSCOPY FOR ASSESSMENT OF LEUKOCYTE-ENDOTHELIAL-INTERACTION IN THE LEFT COLON IN DEXTRAN SULPHATE SODIUM [DSS]-INDUCED COLITIS. S.Farkas. H.Herfarth*, M Steinbaner, M. Anthuber. Department of Surgery, Regensburg, *Department of Internal Med I, Regensburg. Background: Leukocyte adhesion plays a pivotal role in acute and chronic inflammatory bowel disease. Therefore, the objective of this study was to determine leukocyte-endothelial-interaction in intramural collecting venules and submucosal postcapillary venules of the colon by means of in vivo microscopy in DSS induced colitis in mice. Methods Female Balb/c mice, weighing 20 ± 0,3g were used for experiments. Control mice received normal drinking water. Chronic colitis was established after four cycles of feeding 5% DSS -dissolved in their drinking water- for 7 days and normal water for 10 days. Five weeks after the last DSS cycle in vivo microscopy was performed according to the technique by Massberg et al (1) and Gonzalez et al (2) described for small bowel isografts. In anaesthetized mice, at a stable mean arterial pressure above 80 mm Hg, the left colon was mobilized and placed on a specially designed stage. Colonic microcirculatory networks were visualized by i.v. injection of FITC labeled Dextran (2,0pmol/kg). Rhodamin 6G (0,1tamol/kg) was injected, as fluorescent dye to stain leukocytes in vivo. Epi-illumination technique was used with 680 fold magnification. Permanent leukocyte adherence ("sticker") and temporary leukocyte adherence ("roller") were assessed in 10 randomly selected collecting venules and postcapillary venules. Leukocytes adhering longer than 30 sec to the endothelium were determined as stickers (n/mm2 venular surface). Rolling leukocytes were defined as cells moving along the vessel wall with a velocity of less than 30% of the central stream (% of rollers of all free - moving leukocytes during the observation period of 30 see). Results: In mice with chronic colitis we found a significant increase of leukocyte sticking and rolling in submucosal postcapillary (PV) and in intramural collecting venules (CV) compared to controls. Moreover we found an enhanced leukocyte accumulation in postcapillary venules than in collecting venules (p=0,005) in mice with DSS induced colitis.
albC n = 6 alb C + DSS n=6
I sticker in PV sticker in CV (n/mm2 venular surface) 8.3±3.5 2.9±1.8 171.4 ± 29.4*
55.6 ± 13.4"
roller in PV roller in CV (% of all moving leukocytes) 49.7±1.4 42.13±3.8 84.9 ± 3.2*
70.73 ± 4.9*
Mean _+SEM; * = p < 0.01 vs. Balb C Conclusion: We propose a model for assessment of leukocyte-endothelial interaction in the microcirculation of the colon in DSS induced chronic colitis. Leukocyte roiling and sticking in colonic postcapillary and collecting venules is considerably increased in DSS induced chronic colitis, reflecting the degree of inflammation. In addition leukocyte adhesion is most prominent in submucosal postcapillary vessels localized closest to inflammation site. This model may offer a novel approach for quantification of leukocyteendothelial-interaction in experimental inflammatory bowel disease. [1] Massberg et al. Surgery in press 2. Gonzalez et al. Transplantation 1994; (58) 403-8)