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Bile duct ligation and fasting reduce microcirculatory disturbances in indomethacin-induced intestinal inflammation in the rat
April 1995
OWHILE IL-6 mRNA DETECTED BY in situ HYBRIDIZATION IS MORE ABUNDANT IN THE INFLAMED MUCOSA OF CROHN'S DISEASE, IL-8 mRNA EXPRESSION IS ALTERNATIVELY PRONOUNCED IN ULCERATIVE COLITIS. F. ARAI~ T. TAKAHASHI, K. FURUKAWA & H. ASAKURA. Third Department of Internal Medicine, Niigata University School of Medicine. Niigata, JAPAN
Background; Ulcerative colitis(UC) and Crohn's disease(CD) represent two major inflammatory bowel diseases of unknown etiology. Although the enhanced production of interleukin-6(lL-6) and 8(IL-8) at the sites of inflamed intestinal mucosa has recently been indicated by ELISA methods, a little is known about what kinds of cells are mainly responsible for the production of IL-6 and IL-8, especially in vivo. Thus, we tried to demonstrate where these cytokine mRNAs located on mueosal biopsy specimens obtained from patients with UC and CD by means of in situ hybridization. Subjects & Methods; Biopsy specimens were obtained from 10 patients with active UC, 5 patients with CD, 5 patients with inactive UC and 5 normal controls. Tissues were fixed in 4% paraformaldehyde and strored frozen. IL-6 and 8 mRNA were detected by digoxigenin-labeled cDNA probes for human IL-6 and 8 mRNA followed by binding with the mouse monoclonal antibody to digoxigenin. Gold-colloid conjugated anti-mouse antibody and silver particles were employed for the augmented visualization of the specific signals[immunogold silver staining (IGSS) method]. Macrophages(M~) were identified by immunohistochemistry using anti-CD68. Results; The reaction products corresponding to IL-6 mRNA in the inflamed mucosa were found in 2 out of 5 CD patients. None of UC patients and normal controls showed IL-6 expression. In contrast, IL-8 mRNA expression was found in 5 out of 10 active UC patients and in one out of 5 CD patients. None of inactive UC patients and normal controls exhibited IL-8 mRNA. IL-6 or 8 mRNA positive cells in the lamina propria were thought to be Me because of their morphologic appearances and the immunohistochemical reactivity against anti-CD68. These cytokineproducing M~ were found in deeper lamina propria in CD than those in UC, Interestingly, IL-8 mRNA was detected in the mucosal epithelial cell layer in one UC patient. Conelualon; Our data clearly show that enhanced expression of IL-6 by M~ is characteristic in CD, while that of IL-8 in UC, suggesting the alternative participation of each eytokine in the pathogenesis of .UC and CD. Moreover, intestinal epithelial cells in UC also exhibit IL-8 mRNA.
@BILE DUCT LIGATION AND FASTING REDUCE M I C R O C I R C U L A T O R Y D I S T U R B A N C E S IN I N D O M E T H A C I N - I N D U C E D I N T E S T I N A L I N F L A M M A T I O N IN T H E R A T . H. Amdt, K.-D. Palitzsch, J. Sch01merich, and D.N. Granger. Dept. of Physiology; LSU Medical Center, Shreveport, LA, and Dept. of Internal Medicine I, University of Regemburg, 93042 Regensburg, Germany
Indomethacin (INDO) induced intestinal inflammation is preceded by an increase in mucosal permeability and microcireutatory dysfunction. Direct eXposure to luminal bile acids and INDO is necessary for mucosal damage. The objective of this study was m assess the effects of bile duet ligation and fasting on indomethacin induced leukocyte adherence and emigration in rat mesenteric venules. METHODS: In 4 experimental groups a single dose of INDO (7.5 mg/kg, s.c.) was administered 24 hours prior to the experiment. Animals were fed for 6 hours then fasted for 18 hours (3 groups), or fasted for the whole 24 hours (1 group). In 2 of these groups, animals underwent bile duct ligation or sham operation immediately before INDO injection. In 2 control groups, animals received one injection of indomethacin vehicle without or with sham operation prior to application. Ten venules (30 ,urn diameter) per animal were observed using intravital microscopy and the following parameters were monitored: number of adherent and emigrated leukocytes, leukocyte rolling velocity, erythrocyte velocity, venular blood flow, and shear rate. RESULTS: IN'DO alone induced a significant increase in leukocyte adherence and emigration (4.8-fold and 4.2-fold, respectively) compared with untreated animals 24 hours after administration. Sham operation before INDO administration resulted in an even higher rate of adherence and emigration (2.1-fold and 1.8-fold vs. INDO without sham operation, respectively, and 6.4-fold and 6.2-fold vs. sham operation without INDO, respectively). T h e increase after sham operation + INDO was significantly blunted by bile duct ligation (adherence by zlO%, emigration by 55%) to an extent similar to fasting rats after INDO injection (49% and 70%, respectively). Sham operation without INDO resulted in an 1.6-fold increase in leukocyte adhesion without a significant rise in emigration. CONCLUSIONS.: Bile duct ligation and fasting may contribute t o the reduction of indomethacin induced microcirculatory dysfunction by preventing biliary cycling of INDO or by reducing exposure to luminal bile acids.
Immunology, Microbiology, and Inflammatory Disorders A773
• PHENOTYPIC ANALYSIS OF THE INTESTINAL MUCOSAL LYMPHOCYTE POPULATION IN A LYMPHOCYTE TRANSFER MODEL OF INFLAMMATORY BOWEL DISEASE IN SCID MICE. R. Aranda, B. C. Sydora, and M. Kronenberg. Division of Digestive Diseases and Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90024 It has previously been reported that scid mice reconstituted with CD4CD45RB highT lymphocytes from the spleen or lymph node of normal mice develop a wasting disease characterized by chrome intestinal inflammation which is more severe in the colon. In contrast, scid mice that receive the CD4CD45RB I°w lymphocyte population remain unaffected. To determine if there were differences in the number as well as phenotype of the lymphocytes reconstituting the intestinal mueosa of diseased and non-diseased scid recipients, we analyzed the lymphooyte population in the epithelial and lamina propria compartments of both the small and large intestine by flow eytometry. Our analysis revealed the following: 1) CD4CD45RB high cells reconstituted the intestinal mucosa, spleen, mesenterie and peripheral lymph nodes of seid mice whereas CD4CD45RB I°wcells only repopulated the spleen and intestines but not mesenterie or peripheral lymph nodes. 2) In the diseased animals, the number of reconstituting lymphocytes were greater in the small intestine in comparison to the large intestine, although disease was more severe in the colon. 3) Some donor CD4+ cells acquire CD8ctct homodimers in the epithelial compartment These double positive cells were not found in the lamina propria, CD45RB levels decreased following the transfer of the CD4CD45RB high population, whereas CD45RB levels remained low following transfer of the CD4CD45RB l°w population. 4) The reconstituting cells in the intestine express surface markers characteristic of activated mucosal lymphocytes (i.e., ctlEL and CD69). In summary, the data indicate that both pathogenic and nonpathogenie CD4+ T cell populations infiltrate the muoosa and acquire activation markers. Therefore, despite the contrasting abilities of the two populations to cause disease, there appears to be no demonstrable phenotypie difference between the reconstituting lymphocytes present in the intestines of diseased versus non-diseased scid mice.
@LEUKOCYTE-ENDOTHELIAL CELL ADHESION IN INDOMETHACIN-INDUCED INTESTINAL I N F L A M M A T I O N IS C O R R E L A T E D WITH FECAL pH. H. Arndt, K.-D. Palitzsch, J. Sch61merieh. D e p a r t m e n t of I n t e r n a l Medicine [, U n i v e r s i t y of Regensburg, 93042 Regensburg, Germany Recent studies i n d i c a t e that there is a c o r r e l a t i o n between bacterial contents of small bowel and the degree of i n f l a m m a t i o n in various a n i m a l m o d e l s of i n f l a m m a t o r y bowel disease. Variation i n the a m o u n t of different bacterial species m a y shift the pH of intestinal contents. The objective of this s t u d y was to assess the role of small bowel fecal pH on l e u k o c y t e - e n d o t h e l i a l cell i n t e r a c t i o n in a n i n d o m e t h a c i n (INDO)-induced long lasting ileitis in Sprague Dawley rats. METHODS: INDO (7.5 m g / k g s.c.) was injected twice 24 hrs apart. Rats (5 per group) were either fed with the identical diet before and 10 days a f t e r the i n d u c t i o n of i n f l a m m a t i o n until the experiment, or the diet was changed at the time of induction (comb i n a t i o n s of s t a n d a r d diet and special diets: p o o r of fibre; rich in p r o t e i n , u n s a t u r a t e d or s a t u r a t e d fatty acids, or c a r b o h y d r a t e s , p o o r or rich of cholesterol or fat). Ten p o s t c a p i l l a r y m e s e n t e r i c v e n u l e s (30t, m d i a m e t e r l p e r animal were o b s e r v e d using intravital m i c r o s c o p y , a n d the following m i c r o c i r c u l a t o r y p a r a m e t e r s were m o n i t o r e d : n u m b e r of a d h e r e n t and e m i g r a t e d leukocytes, l e u k o c y t e r o l l i n g velocity, e r y t h r o c v t e velocity, v e n u l a r blood flow, arid shear rate. Fecal pH of certain secg tions of the small bowel was d e t e r m i n e d in I : I 0 solution with 0.9% NaC1. RESULTS: The e° a p p l i c a t i o n of v a r i o u s diets or diet combia,3 n a t i o n s r e s u l t e d in fecal p H - d i f f e r e n c e s ranging from 7.8 to 8.8 in the lower section ~38-~ ~ • • °ftheileum'Differentdi~tsresultedin a n *= ~' J < ~ , ~" • in- o r d e c r e a s e in l e u k o c y t e a d h e r e n c e , e m i g r a t i o n a n d rolling velc3citv c o m p a r e d ~3,3 with o u r c o n t r o l ( s t a n d a r d ) , While s h e a r -~2,s rate was n e a r l y unaffected. There was a ~ / n e g a t i v e correl~.tion between fecal pH and x~ leukocyte a d h e r e n c e (figure, x=-0.65y+10.3, ~ , 3 r= 0.70), but n o t with o t h e r m i c r o c i r c u l a tory p a r a m e t e r s , CONCLUSIONS: Modulating 1 ; , the pH of feces in the s m a l l bowel by ~,s • • d i f f e r e n t d i e t s c h a n g e s the INDOqnduced l e u k o c y t e - e n d o t h e l i a l cell adhesion. These 1,3 d a t a m a y p r o v i d e a r a t i o n a l e for the 7,7 8,2 8,7 t h e r a p e u t i c effect of e n t e r a l n u t r i t i o n in FECALpH Crohn" s disease.
4,8~. 1
OWHILE IL-6 mRNA DETECTED BY in situ HYBRIDIZATION IS MORE ABUNDANT IN THE INFLAMED MUCOSA OF CROHN'S DISEASE, IL-8 mRNA EXPRESSION IS ALTERNATIVELY PRONOUNCED IN ULCERATIVE COLITIS. F. ARAI~ T. TAKAHASHI, K. FURUKAWA & H. ASAKURA. Third Department of Internal Medicine, Niigata University School of Medicine. Niigata, JAPAN
Background; Ulcerative colitis(UC) and Crohn's disease(CD) represent two major inflammatory bowel diseases of unknown etiology. Although the enhanced production of interleukin-6(lL-6) and 8(IL-8) at the sites of inflamed intestinal mucosa has recently been indicated by ELISA methods, a little is known about what kinds of cells are mainly responsible for the production of IL-6 and IL-8, especially in vivo. Thus, we tried to demonstrate where these cytokine mRNAs located on mueosal biopsy specimens obtained from patients with UC and CD by means of in situ hybridization. Subjects & Methods; Biopsy specimens were obtained from 10 patients with active UC, 5 patients with CD, 5 patients with inactive UC and 5 normal controls. Tissues were fixed in 4% paraformaldehyde and strored frozen. IL-6 and 8 mRNA were detected by digoxigenin-labeled cDNA probes for human IL-6 and 8 mRNA followed by binding with the mouse monoclonal antibody to digoxigenin. Gold-colloid conjugated anti-mouse antibody and silver particles were employed for the augmented visualization of the specific signals[immunogold silver staining (IGSS) method]. Macrophages(M~) were identified by immunohistochemistry using anti-CD68. Results; The reaction products corresponding to IL-6 mRNA in the inflamed mucosa were found in 2 out of 5 CD patients. None of UC patients and normal controls showed IL-6 expression. In contrast, IL-8 mRNA expression was found in 5 out of 10 active UC patients and in one out of 5 CD patients. None of inactive UC patients and normal controls exhibited IL-8 mRNA. IL-6 or 8 mRNA positive cells in the lamina propria were thought to be Me because of their morphologic appearances and the immunohistochemical reactivity against anti-CD68. These cytokineproducing M~ were found in deeper lamina propria in CD than those in UC, Interestingly, IL-8 mRNA was detected in the mucosal epithelial cell layer in one UC patient. Conelualon; Our data clearly show that enhanced expression of IL-6 by M~ is characteristic in CD, while that of IL-8 in UC, suggesting the alternative participation of each eytokine in the pathogenesis of .UC and CD. Moreover, intestinal epithelial cells in UC also exhibit IL-8 mRNA.
@BILE DUCT LIGATION AND FASTING REDUCE M I C R O C I R C U L A T O R Y D I S T U R B A N C E S IN I N D O M E T H A C I N - I N D U C E D I N T E S T I N A L I N F L A M M A T I O N IN T H E R A T . H. Amdt, K.-D. Palitzsch, J. Sch01merich, and D.N. Granger. Dept. of Physiology; LSU Medical Center, Shreveport, LA, and Dept. of Internal Medicine I, University of Regemburg, 93042 Regensburg, Germany
Indomethacin (INDO) induced intestinal inflammation is preceded by an increase in mucosal permeability and microcireutatory dysfunction. Direct eXposure to luminal bile acids and INDO is necessary for mucosal damage. The objective of this study was m assess the effects of bile duet ligation and fasting on indomethacin induced leukocyte adherence and emigration in rat mesenteric venules. METHODS: In 4 experimental groups a single dose of INDO (7.5 mg/kg, s.c.) was administered 24 hours prior to the experiment. Animals were fed for 6 hours then fasted for 18 hours (3 groups), or fasted for the whole 24 hours (1 group). In 2 of these groups, animals underwent bile duct ligation or sham operation immediately before INDO injection. In 2 control groups, animals received one injection of indomethacin vehicle without or with sham operation prior to application. Ten venules (30 ,urn diameter) per animal were observed using intravital microscopy and the following parameters were monitored: number of adherent and emigrated leukocytes, leukocyte rolling velocity, erythrocyte velocity, venular blood flow, and shear rate. RESULTS: IN'DO alone induced a significant increase in leukocyte adherence and emigration (4.8-fold and 4.2-fold, respectively) compared with untreated animals 24 hours after administration. Sham operation before INDO administration resulted in an even higher rate of adherence and emigration (2.1-fold and 1.8-fold vs. INDO without sham operation, respectively, and 6.4-fold and 6.2-fold vs. sham operation without INDO, respectively). T h e increase after sham operation + INDO was significantly blunted by bile duct ligation (adherence by zlO%, emigration by 55%) to an extent similar to fasting rats after INDO injection (49% and 70%, respectively). Sham operation without INDO resulted in an 1.6-fold increase in leukocyte adhesion without a significant rise in emigration. CONCLUSIONS.: Bile duct ligation and fasting may contribute t o the reduction of indomethacin induced microcirculatory dysfunction by preventing biliary cycling of INDO or by reducing exposure to luminal bile acids.
Immunology, Microbiology, and Inflammatory Disorders A773
• PHENOTYPIC ANALYSIS OF THE INTESTINAL MUCOSAL LYMPHOCYTE POPULATION IN A LYMPHOCYTE TRANSFER MODEL OF INFLAMMATORY BOWEL DISEASE IN SCID MICE. R. Aranda, B. C. Sydora, and M. Kronenberg. Division of Digestive Diseases and Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90024 It has previously been reported that scid mice reconstituted with CD4CD45RB highT lymphocytes from the spleen or lymph node of normal mice develop a wasting disease characterized by chrome intestinal inflammation which is more severe in the colon. In contrast, scid mice that receive the CD4CD45RB I°w lymphocyte population remain unaffected. To determine if there were differences in the number as well as phenotype of the lymphocytes reconstituting the intestinal mueosa of diseased and non-diseased scid recipients, we analyzed the lymphooyte population in the epithelial and lamina propria compartments of both the small and large intestine by flow eytometry. Our analysis revealed the following: 1) CD4CD45RB high cells reconstituted the intestinal mucosa, spleen, mesenterie and peripheral lymph nodes of seid mice whereas CD4CD45RB I°wcells only repopulated the spleen and intestines but not mesenterie or peripheral lymph nodes. 2) In the diseased animals, the number of reconstituting lymphocytes were greater in the small intestine in comparison to the large intestine, although disease was more severe in the colon. 3) Some donor CD4+ cells acquire CD8ctct homodimers in the epithelial compartment These double positive cells were not found in the lamina propria, CD45RB levels decreased following the transfer of the CD4CD45RB high population, whereas CD45RB levels remained low following transfer of the CD4CD45RB l°w population. 4) The reconstituting cells in the intestine express surface markers characteristic of activated mucosal lymphocytes (i.e., ctlEL and CD69). In summary, the data indicate that both pathogenic and nonpathogenie CD4+ T cell populations infiltrate the muoosa and acquire activation markers. Therefore, despite the contrasting abilities of the two populations to cause disease, there appears to be no demonstrable phenotypie difference between the reconstituting lymphocytes present in the intestines of diseased versus non-diseased scid mice.
@LEUKOCYTE-ENDOTHELIAL CELL ADHESION IN INDOMETHACIN-INDUCED INTESTINAL I N F L A M M A T I O N IS C O R R E L A T E D WITH FECAL pH. H. Arndt, K.-D. Palitzsch, J. Sch61merieh. D e p a r t m e n t of I n t e r n a l Medicine [, U n i v e r s i t y of Regensburg, 93042 Regensburg, Germany Recent studies i n d i c a t e that there is a c o r r e l a t i o n between bacterial contents of small bowel and the degree of i n f l a m m a t i o n in various a n i m a l m o d e l s of i n f l a m m a t o r y bowel disease. Variation i n the a m o u n t of different bacterial species m a y shift the pH of intestinal contents. The objective of this s t u d y was to assess the role of small bowel fecal pH on l e u k o c y t e - e n d o t h e l i a l cell i n t e r a c t i o n in a n i n d o m e t h a c i n (INDO)-induced long lasting ileitis in Sprague Dawley rats. METHODS: INDO (7.5 m g / k g s.c.) was injected twice 24 hrs apart. Rats (5 per group) were either fed with the identical diet before and 10 days a f t e r the i n d u c t i o n of i n f l a m m a t i o n until the experiment, or the diet was changed at the time of induction (comb i n a t i o n s of s t a n d a r d diet and special diets: p o o r of fibre; rich in p r o t e i n , u n s a t u r a t e d or s a t u r a t e d fatty acids, or c a r b o h y d r a t e s , p o o r or rich of cholesterol or fat). Ten p o s t c a p i l l a r y m e s e n t e r i c v e n u l e s (30t, m d i a m e t e r l p e r animal were o b s e r v e d using intravital m i c r o s c o p y , a n d the following m i c r o c i r c u l a t o r y p a r a m e t e r s were m o n i t o r e d : n u m b e r of a d h e r e n t and e m i g r a t e d leukocytes, l e u k o c y t e r o l l i n g velocity, e r y t h r o c v t e velocity, v e n u l a r blood flow, arid shear rate. Fecal pH of certain secg tions of the small bowel was d e t e r m i n e d in I : I 0 solution with 0.9% NaC1. RESULTS: The e° a p p l i c a t i o n of v a r i o u s diets or diet combia,3 n a t i o n s r e s u l t e d in fecal p H - d i f f e r e n c e s ranging from 7.8 to 8.8 in the lower section ~38-~ ~ • • °ftheileum'Differentdi~tsresultedin a n *= ~' J < ~ , ~" • in- o r d e c r e a s e in l e u k o c y t e a d h e r e n c e , e m i g r a t i o n a n d rolling velc3citv c o m p a r e d ~3,3 with o u r c o n t r o l ( s t a n d a r d ) , While s h e a r -~2,s rate was n e a r l y unaffected. There was a ~ / n e g a t i v e correl~.tion between fecal pH and x~ leukocyte a d h e r e n c e (figure, x=-0.65y+10.3, ~ , 3 r= 0.70), but n o t with o t h e r m i c r o c i r c u l a tory p a r a m e t e r s , CONCLUSIONS: Modulating 1 ; , the pH of feces in the s m a l l bowel by ~,s • • d i f f e r e n t d i e t s c h a n g e s the INDOqnduced l e u k o c y t e - e n d o t h e l i a l cell adhesion. These 1,3 d a t a m a y p r o v i d e a r a t i o n a l e for the 7,7 8,2 8,7 t h e r a p e u t i c effect of e n t e r a l n u t r i t i o n in FECALpH Crohn" s disease.
4,8~. 1