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Inflammatory bowel disease in IL-2 deficient mice requires T, but not B lymphocytes
April 1995
Immunology, Microbiology, and Inflammatory Disorders A867
• INFLAMMATORY BOWEL DISEASE IN IL-2 DEFICIENT MICE REQUIRES T, BUT NOT B LYMPHOCYTES A. Ma, M.W. Datta, E. Margoslan, I. Horak, and F.W. Ale Center for Blood Research, Harvard Medical School, Boston, MA Howard Hughes Medical Institute, ChildrenTs Hospital, Boston, MA Pur__~: To determine the roles of particular lymphocyte populations and autoantlbodies in the development of colonic inflammation in IL-2 deficient (IL-2 -/-) mice. Methods: We have bred IL-2- - mice to two strains of immunodeficient mice generated by gene targeting in our laboratory: JH deficient (JH_I_) mice lacking mature B cells and RAG-2 deficient (RAG-2 " ) mice lacking both mature B end T cells. We have then observed and scored single and double mutant mice for the dev~lopment of eol?nlc inflammation. Results: RAG-2-'- mlca and J~ " mice do not develop bowel inflammation in our colony when observed for over on~/!ear. The intestinal mucosal inflamm~eion observed in IL-2 mice is essentially absent in IL-2-'-/RAG-2- - double mutant mice, confirming that mature lymphocytes are required for thy_ manifestation of clinical disease. In contrast, IL-2 / JH- - double mutant mice develop the same clinical and histological intestinal disease (including lymphocyte predominant lamina propria and intra-epithelial inflammation, crypt ab~£ess formation and epithelial erosion) as is seen in IL-2 mice. Conclusions: While environmental pathogens probably trigge~ a~n~rlate immune response and cause disease in IL-2-'mice, they do not directly cause disease. Mature T but not B lymphocytes (or their autoantlbodies) are required for bowel inflammation in IL-2 mice.
• COLITIS PATIENTS NOT RESPONDING TO HIGH DOSE CORTICOSTEROIDS HAVE A LOW GLUCOCORTICOID RECEPTOR CONTENT OF THEIR MONONUCLEAR CELLS. G.S. Madretsma. A.P.M. van Dijk, J.H.P. Wilson, F.J. Zijlstra. Depts. of Pharmacology and Internal Medicine, Erasmus University, Rotterdam, The Netherlands. A minority of patients with inflammatory bowel disease (IBD) do not respond to high doses of glucocorticosteroids (GC), Variation in anti-inflammatory response to cortico-steroids could be due to variation in the number of the glucocorticoid receptors (GR) in mononuclear cells (MNC). In order to test this hypothesis we assessed the number of GR of these cells in three groups: healthy controls, IBD-patients who had responded to GC-treatment (responders) and IBD-patients who had undergone colectomy because their colitis failed to respond to high dose GC (non-responders). MNC were isolated from heparinized peripheral blood from each individual and GR number was determined by means of a whole cell binding assay with tritiated dexamethasone. Samples were analyzed in a blinded fashion. Results are expressed as mean _.+ SEM GR number per cell Healthy controls (n=9): 4430 _+ 340 Responders (n=6): 3900 - 210 Non-responders (n=6): 2450 _+ 310 * Patients who do not respond to treatment with high dose GC have a significantly lower number of GR in their MNC in comparison to responders and healthy controls (* p<0.01). Responders do not differ from healthy controls as far as GR content is concerned. These findings strongly suggest a correlation between GR number in MNC and the effectiveness of corticosteroid treatment in IBD. Supported by Foundation.
the
Netherlands
Digestive
Diseases
• C Y T O S K E L E T A L R E G U L A T I O N OF CACO-2 INTESTINAL M O N O L A Y E R PARACELLULAR PERMEABILITY. Thomas Y. Ma, Daniel Hollander, Don Nguyen, Nell I-Ioa. Division of Gastroenterology, Department of Medicine, DVA Medical Center, Long Beach, California and University of California, Irvine, California
Intestinal permeability is increased in Crohn's disease. Recent evidence suggests that abnormal increase in intestinal permeability may be an important etiologic factor in Crohn's disease and in NSAID - associated enteropathy. Despite the potential importance of paracellular pathways in the pathogenesis of these diseases, the intracellular factors and processes which regulate and cause alteration of intestinal paracellular permeability are not well understood. The purpose of this study was to examine some of the intracellular processes involved in cytoskeletal regulation of intestinal epithelial paracellular permeability using the filter-grown Caco-2 intestinal epithelial rnonolayers. Cytochalasin-b and colchicine were used to disrupt the cytoskeletal elements, actin microfilaments and microtubules. Cytochalasin-h (5 I.tg/ml) and colchicine (2 x 10-5M) at the doses used caused marked depolyrnerization and disruption of actin inicrofilaments and microtubules, respectively. Cytochalasin-b-induced disruption of actin microfilaments resulted in perturbation of tight junctions and desmosomes and an increase in Caco-2 monolayer paracellular permeability. The cytochalasin-b-induced disruption of actin microfilaments and subsequent changes in intercellular junctional complexes and paracelhilar permeability were not affected by inhibitors of protein synthesis (actinomycin-D or cycloheximide) or microtubule function (colchicine), but were inhibited by metabolic energy inhibitors (2,4-dinitrophenol or sodium azide). The cytochalasin-b-induced disturbance in Caco-2 actin microfilaments and intercellular junctional complexes and increase in paracellular permeability were rapidly reversed. The paracellular pathway "re-tightening" following cytochalasin-b removal was not affected by actinomycin-D, cycloheximide, or colchicine, but was inhibited by 2,4-dinitrophenol and sodium azide. The colchicine-induced disruption of microtubules did not have significant effect on actin microfilaments, intercelhilar junctions, or paracellular permeability. These findings suggest that cytochalasin-b-induced increase in Caco-2 monolayer paracellular permeability was due to actin rnicrofilament mediated perturbation of intercellular junctional complexes. The re-tightening of paracellular pathways (following removal of cytochalasin-b) resulted from energy-mediated re-assembly of pre-existing actin microfilaments and intercellular junctional complexes; this re-closure process did not require protein synthesis or microtubule-mediated shuttling process.
• NICOTINE INHIBITS THE PRODUCTION OF IL-2 AND TNF-¢ BY PERIPHERAL BLOOD AND INTESTINAL MONONUCLEAR CELLS. G,S.Madretsma, A.P.M.van Dijk, C.J.AM.Tak, G.J.Donze, J.H.P.Wilson, F.J.Zijlstra. Depts. of Pharmacology and Internal Medicine, Erasmus University, Rotterdam, The Netherlands. Smoking protects against ulcerative coliti: (UC), and nicotine patches have a beneficial symptomatJ,~ effect in patients with UC. To find an explanation for this re ~ponse to nicotine in UC, we looked for effects of nicotine or cytokine production by mononuclear cells (MNC) and for nicotine (NIC) receptors on MNC. MNC were isolate1 from peripheral blood of healthy volunteers and from .,~urgical specimens from patients undergoing colectomy for cancer. Segments used to isolate MNC were taken at least 5 cm from the cancer. Non-adherent MNC were preincubated with varying concentrations of nicotine for 24 hours, 10pg/ml of phythemagglutinin was then added and the MNC incubated for another 24 hrs. Additional cells were used for receptor binding studies. Nicotine significantly inhibited the production of IL-2 and TNF-~z by the non-adherent MNC in a dose-dependent fashion in the range of 10~ to 10.7 M, with a maximum inhibition of 51% and 48% respectively. This inhibitory effect could not be antagonized by the NIC1 receptor antagonist, hexamethonium, or the NIC2 receptor antagonist, pancuronium. The MNC had 2420 _+ 360 NIC receptors per cell. These NIO-receptors had no affinity for hexamethonium or pancuronium, indicating a non-cholinergic nature for the receptor. Nicotine inhibits the production of IL-2 and TNF-~ by peripheral blood and colon mucosal mononuclear cells, via a non-cholinergic nicotine receptor. The suppression of IL-2 and TNF-~z provides an explanation for the beneficial effects of smoking and nicotine in ulcerative colitis.
Immunology, Microbiology, and Inflammatory Disorders A867
• INFLAMMATORY BOWEL DISEASE IN IL-2 DEFICIENT MICE REQUIRES T, BUT NOT B LYMPHOCYTES A. Ma, M.W. Datta, E. Margoslan, I. Horak, and F.W. Ale Center for Blood Research, Harvard Medical School, Boston, MA Howard Hughes Medical Institute, ChildrenTs Hospital, Boston, MA Pur__~: To determine the roles of particular lymphocyte populations and autoantlbodies in the development of colonic inflammation in IL-2 deficient (IL-2 -/-) mice. Methods: We have bred IL-2- - mice to two strains of immunodeficient mice generated by gene targeting in our laboratory: JH deficient (JH_I_) mice lacking mature B cells and RAG-2 deficient (RAG-2 " ) mice lacking both mature B end T cells. We have then observed and scored single and double mutant mice for the dev~lopment of eol?nlc inflammation. Results: RAG-2-'- mlca and J~ " mice do not develop bowel inflammation in our colony when observed for over on~/!ear. The intestinal mucosal inflamm~eion observed in IL-2 mice is essentially absent in IL-2-'-/RAG-2- - double mutant mice, confirming that mature lymphocytes are required for thy_ manifestation of clinical disease. In contrast, IL-2 / JH- - double mutant mice develop the same clinical and histological intestinal disease (including lymphocyte predominant lamina propria and intra-epithelial inflammation, crypt ab~£ess formation and epithelial erosion) as is seen in IL-2 mice. Conclusions: While environmental pathogens probably trigge~ a~n~rlate immune response and cause disease in IL-2-'mice, they do not directly cause disease. Mature T but not B lymphocytes (or their autoantlbodies) are required for bowel inflammation in IL-2 mice.
• COLITIS PATIENTS NOT RESPONDING TO HIGH DOSE CORTICOSTEROIDS HAVE A LOW GLUCOCORTICOID RECEPTOR CONTENT OF THEIR MONONUCLEAR CELLS. G.S. Madretsma. A.P.M. van Dijk, J.H.P. Wilson, F.J. Zijlstra. Depts. of Pharmacology and Internal Medicine, Erasmus University, Rotterdam, The Netherlands. A minority of patients with inflammatory bowel disease (IBD) do not respond to high doses of glucocorticosteroids (GC), Variation in anti-inflammatory response to cortico-steroids could be due to variation in the number of the glucocorticoid receptors (GR) in mononuclear cells (MNC). In order to test this hypothesis we assessed the number of GR of these cells in three groups: healthy controls, IBD-patients who had responded to GC-treatment (responders) and IBD-patients who had undergone colectomy because their colitis failed to respond to high dose GC (non-responders). MNC were isolated from heparinized peripheral blood from each individual and GR number was determined by means of a whole cell binding assay with tritiated dexamethasone. Samples were analyzed in a blinded fashion. Results are expressed as mean _.+ SEM GR number per cell Healthy controls (n=9): 4430 _+ 340 Responders (n=6): 3900 - 210 Non-responders (n=6): 2450 _+ 310 * Patients who do not respond to treatment with high dose GC have a significantly lower number of GR in their MNC in comparison to responders and healthy controls (* p<0.01). Responders do not differ from healthy controls as far as GR content is concerned. These findings strongly suggest a correlation between GR number in MNC and the effectiveness of corticosteroid treatment in IBD. Supported by Foundation.
the
Netherlands
Digestive
Diseases
• C Y T O S K E L E T A L R E G U L A T I O N OF CACO-2 INTESTINAL M O N O L A Y E R PARACELLULAR PERMEABILITY. Thomas Y. Ma, Daniel Hollander, Don Nguyen, Nell I-Ioa. Division of Gastroenterology, Department of Medicine, DVA Medical Center, Long Beach, California and University of California, Irvine, California
Intestinal permeability is increased in Crohn's disease. Recent evidence suggests that abnormal increase in intestinal permeability may be an important etiologic factor in Crohn's disease and in NSAID - associated enteropathy. Despite the potential importance of paracellular pathways in the pathogenesis of these diseases, the intracellular factors and processes which regulate and cause alteration of intestinal paracellular permeability are not well understood. The purpose of this study was to examine some of the intracellular processes involved in cytoskeletal regulation of intestinal epithelial paracellular permeability using the filter-grown Caco-2 intestinal epithelial rnonolayers. Cytochalasin-b and colchicine were used to disrupt the cytoskeletal elements, actin microfilaments and microtubules. Cytochalasin-h (5 I.tg/ml) and colchicine (2 x 10-5M) at the doses used caused marked depolyrnerization and disruption of actin inicrofilaments and microtubules, respectively. Cytochalasin-b-induced disruption of actin microfilaments resulted in perturbation of tight junctions and desmosomes and an increase in Caco-2 monolayer paracellular permeability. The cytochalasin-b-induced disruption of actin microfilaments and subsequent changes in intercellular junctional complexes and paracelhilar permeability were not affected by inhibitors of protein synthesis (actinomycin-D or cycloheximide) or microtubule function (colchicine), but were inhibited by metabolic energy inhibitors (2,4-dinitrophenol or sodium azide). The cytochalasin-b-induced disturbance in Caco-2 actin microfilaments and intercellular junctional complexes and increase in paracellular permeability were rapidly reversed. The paracellular pathway "re-tightening" following cytochalasin-b removal was not affected by actinomycin-D, cycloheximide, or colchicine, but was inhibited by 2,4-dinitrophenol and sodium azide. The colchicine-induced disruption of microtubules did not have significant effect on actin microfilaments, intercelhilar junctions, or paracellular permeability. These findings suggest that cytochalasin-b-induced increase in Caco-2 monolayer paracellular permeability was due to actin rnicrofilament mediated perturbation of intercellular junctional complexes. The re-tightening of paracellular pathways (following removal of cytochalasin-b) resulted from energy-mediated re-assembly of pre-existing actin microfilaments and intercellular junctional complexes; this re-closure process did not require protein synthesis or microtubule-mediated shuttling process.
• NICOTINE INHIBITS THE PRODUCTION OF IL-2 AND TNF-¢ BY PERIPHERAL BLOOD AND INTESTINAL MONONUCLEAR CELLS. G,S.Madretsma, A.P.M.van Dijk, C.J.AM.Tak, G.J.Donze, J.H.P.Wilson, F.J.Zijlstra. Depts. of Pharmacology and Internal Medicine, Erasmus University, Rotterdam, The Netherlands. Smoking protects against ulcerative coliti: (UC), and nicotine patches have a beneficial symptomatJ,~ effect in patients with UC. To find an explanation for this re ~ponse to nicotine in UC, we looked for effects of nicotine or cytokine production by mononuclear cells (MNC) and for nicotine (NIC) receptors on MNC. MNC were isolate1 from peripheral blood of healthy volunteers and from .,~urgical specimens from patients undergoing colectomy for cancer. Segments used to isolate MNC were taken at least 5 cm from the cancer. Non-adherent MNC were preincubated with varying concentrations of nicotine for 24 hours, 10pg/ml of phythemagglutinin was then added and the MNC incubated for another 24 hrs. Additional cells were used for receptor binding studies. Nicotine significantly inhibited the production of IL-2 and TNF-~z by the non-adherent MNC in a dose-dependent fashion in the range of 10~ to 10.7 M, with a maximum inhibition of 51% and 48% respectively. This inhibitory effect could not be antagonized by the NIC1 receptor antagonist, hexamethonium, or the NIC2 receptor antagonist, pancuronium. The MNC had 2420 _+ 360 NIC receptors per cell. These NIO-receptors had no affinity for hexamethonium or pancuronium, indicating a non-cholinergic nature for the receptor. Nicotine inhibits the production of IL-2 and TNF-~ by peripheral blood and colon mucosal mononuclear cells, via a non-cholinergic nicotine receptor. The suppression of IL-2 and TNF-~z provides an explanation for the beneficial effects of smoking and nicotine in ulcerative colitis.