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TNBS-induced colitis in rats can be transferred by T cells
April 1995
• MUCOSAL IMMUNOSUPPRESSION BY INDUCTION OF APOPTOSIS IN LAMINA PROPRIA L Y M P t t O C Y T E S G.C. O~Sullivan, J.K. Collins, D. Collins, A. O'Mahony, and F. Shanahan. University College Cork, IRELAND. Therapeutic exploitation of a mechanism for selective suppression of activat©d lymphoeytes is desireable in several disorders. We previously desoribed and partially purified a factor produced by human esophageal squamous cell carcinomas that induces lmmunosuppression in vivo and in vitro (J Immunol 1993; 151:4847-56; Gastroenterology 1994; 106: A424). The tumor-derived factor induces apoptosis in peripheral blood lymphoeytes (PBL) but only when they are preactivated. Since normal intestinal lamina opria lymphoeytes (LPL) represent an activated immune compartment, we ve determined the activity of the tumor-derived immune suppressor factor (ISF) on human and marine LPL and examined its mechanism of action. The ISF was found to exert profound, dose-dependent, inhibition of resting and PHA-stimulated LPL proliferation (thymidine incorporation). When background apoptosis was controlled for (which was significantly greater in LPL than PBL from the same patient), the ISF induced significanl apoptosis as shown by morphology, flow cytometry, (propidium iodide staining with gating on eytokeratin-negative coils), and DNA gel eleetrophoresis. Because surface apo-1/FAS is an endogenous trigger mechanism of physiologic apoptosis, we determined whether ISF works through this pathway. To do this, we used PBL and LPL from MRL Ipr/lpr mice, which have a deficiency of functional apo-1/FAS receptor, and compared results with the MRL congenic control. The ISF was acuve on both FAS-mutant and FAS-normal lymphoeytes, indicating that it does not require an intact FAS signal pathway for apeptosis. To further clarify its mechanism of action, we measured the intraceUular cyclic AMP (cAMP) response to ISF in resting and activated lymphocytas, cAMP is negative regulator of activation and proliferation in lymphocytes. ISF induced significant elevation of cAMP levels to greater than 200pg/106 cells in activated lymphocytes, but resting lymphocytes did not respond to ISF with cAMP levels remaining at less than 20pg/106 cells, whereas both resting and activated cells responded to forskolin. In conclusion, the ISF of esophageal squamous cancer suppresses lymphocyte proliferation and induces apoptosis by increasing intracellular cAMP. The factor works through a pathway other than apo-I/FAS that appears to be expressed only in activated lymphocytes. Because LPL exist in a state of relative activation, they are exquisitely sensitive to this agent
which may ultimatelyhave therapeuticpotential.
• TNBS-INDUCED COLITIS IN RATS CAN BE TRANSFERRED BY T CELLS. MJHJ Palmen ~, OLC Wijburg ~, AS Pefiaz, SGM Meuwissen2, EP van Rees ~. Dept. of Cell Biology/Immunology ~ and Dept. of Gastroenterology 2, Faculty of Medicine, Vrije Universiteit, Amsterdam, The Netherlands. Introduction: Colitis can be induced in rats by intracolonic administration of 2,4,6-trinitrobenzene sulphonic acid (TNBS) in ethanol. As second administration of TNBS (subcutaneously) results in a prolonged chronic phase of inflammation, the rats probably have been sensitized by the first dose of TNBS, given in the colon (Palmen et al, Clin Exp Immunol 1995, in press). This suggests that T cells play an important role in the pathogenesis of experimental colitis. Therefore, the aim of the present study was to examine if T cell transfer from TNBS/ethanol sensitized rats induces colitis in naive recipients. Methods: Colitis was induced by a single rectal administration of 30 mg TNBS in ethanol 30%. Seventeen days later, T cells were isolated from the spleens and a high dose was administered intravenously to naive rats. As controls we administered T ceils of normal healthy rats and splenic leukocytes without T ceils to naive recipients. After 2 hours, either TNBS or saline was given intraperitoneally to the recipients. Three days later, the animals were sacrificed, macroscopic damage scores of the colon were established and macrophages, granulocytes and lymphocytes were studied in situ in the colon and small intestine using immunohistochemistry. Quantification was done using the IBAS image analysis system. Results: Edema was found in the colon of the recipients of T-cells obtained from TNBStreated rats, but not in the controls. An influx of granulocytes and macrophages was observed only in the colonic submucosa of recipients of sensitized T cells, which suggests that the animals suffer from an acute inflammation in the colon. We also observed an increase in the number of T cells in the colon. No effect was found in control rats. To examine which subset of T cells caused this effect, CD4+ T ceils were transferred, but this did not result in macroscopic or microscopic signs of inflammation. Conclusion: Colitis can be transferred to naive rats by T cells, obtained from rats with TNBS-ethanol induced experimental colitis. It might be possible that T cells recognize, CD8 involved, a specific protein in the colon in combination with TNBS.
Immunology, Microbiology, and Inflammatory Disorders A889
BENEFICIAL EFFECT OF APOCYNIN, A PLANT-DERIVED NADPH OXIDASE ANTAGONIST, IN TNBS-INDUCED EXPERIMENTAL COLITIS. MJHJ Palmen ~, CJ Beukelman3, RGM Mooij ~, SGM Meuwissen2, EP van ReesI. ~Dept of Cell Biology/Immunology, 2Dept of Gastroenterology, Fac. of Medicine, Vrije Universiteit, Amsterdam; ~Dept. of Pharmacognosy, Fac. Pharmacy, Universiteit Utrecht, The Netherlands. Introduction: Reactive oxygen species (ROS) produced by activated neutrophils and macrophages may play an important role in tissue damage in inflammatory bowel disease (IBD), The plantphenol 4-hydroxy-3-meth0xyacetophenone (trivial name: apocynin), an NADPH oxidase inhibitor, is newly isolated from traditional anti-inflammatory medicinal herb Picrorhiza kurroa. In the present study, the effect of apocynin in a rat model for IBD was studied. Methods: Colitis was induced in Wistar rats on day 0 by a single rectal administration of 30 mg TNBS in ethanol 30%. Rats were treated on day 0 and day 3 with 4 mg/kg apocynin i.v. Controls received saline i.v. in equal volumes. Rats were sacrificed on day 7 and macroscopical damage scores of the colon were determined on a 0-6 scale. Macrophages and granulocytes in the colon were studied in situ by immunohistochemistry and were quantified using the IBAS image analysis system. Furthermore, MPO activity was determined in colonic tissue, Results: Mean damage scores of the colon were significantly improved in the apocynin treated rats compared to the controls (mean damage score controls 3.4, mean damage scores after apocynin treatment 1). MPO activity was decreased with 98% in apocynin treated rats. Strikingly, a decrease was found in the number of granulocytes (67%) and macrophages (68%) in the colon. Conclusion: Apocynin has a striking beneficial effect on experimental colitis. The results suggest that treatment with apocynin, in addition to inhibiting ROS production, also decreases the influx of monocytes and neutrophils into the tissue.
ICAM-1 EXPRESSION IN THE SPLANCHNIC ORGANS IN VIVO~ EFFECTS OF ENDOTOXIN. J Pands, MA Perry#, A. Manning*, JM Russell, DC Anderson*, DN Granger. Dept Physiology. LSU Medical Center, Shreveport, LA 71111.
#Universitty of Soutn Wales, Sidney, Australia. *Upjohn Laboratories, Kalamazoo, M149001. ICAM-1 has been implicated in the recruitment of leukocytes into inflamed regions of the GI tract. However, there is no quantitative data in the literature that addressed the in vivo responses of this endothelial cell adhesion molecule to an inflammatory stimulus. The objectives of this study were to characterize and compare the constitutive and inducible expression of ICAM- 1 in splanchnic organs after exposure to endotoxin. Methods: ICAM- 1 expression was studied in the splanchnic organs of untreated or endotoxin-treated rats, at 3, 5, 9, 12, and 24 hours after endotoxin administration (5 mg/Kg, i.p.). ICAM-1 expression was measured using 125Ilabeled anti,rat ICAM-1 mAb (1A29), and an isotype matched control mAb: ~3~IP-23 (anti-dog ICAM-1) to correct for nonspecific accumulation of 1A29.ICAM-1 corrected activity was expressed as (% injected activity ~2sI-ICAM-1/g)- (% injected activity lzXI-P23/g). ICAM- 1 mRNA was measured in the intestine by Northern blot analysis. Results: ICAM-1 mAb binding was observed in all splanchnic organs. ~zsI-ICAM-1 mAb accumulation was blocked dose-dependently by increasing doses of cold ICAM- 1 mAb, whereas a non-binding mAb had no effect. The accumulation of ICAM-1 mAb varied widely in the different organs. It was high in the liver (4.4_-L-0.7%/g), spleen (4.2_+0.4%/g) and kidneys (1.3--+0.2 %/g), and much lower in stomach (0.06_+0.006 %/g) small bowel (0.15!-0.02) large bowel (0.14_+0:02 %/g), pancreas (0.09-20.01%/g) and mesentery (0.11_+0.01). Endotoxin induced an increase in ICAM- 1 mAb binding in the organs with a low basal expression: stomach (4-fold), and small intestine, large intestine, pancreas, and mesentery (3-fold), with no changes observed in liver, and. Maximal expression occurred at 9-12 hours after endotoxin administration, and persisted at 24 h. ICAM-1 mRNA was not detected in the small bowel under basal conditions, but a marked increase occurred at 3 h, i.e., preceding the significant increase in ICAM-1 expression at 5 h; Conclusions: ICAM-1 is constitutively expressed in all splanchnic organs in the rat_ The endothefial cell adhesion molecule is upregulated by endotoxin in those tissues with a low constitutive expression. Maximal upregulation occurs at 9-12 hours after the inflammatory stimulus. (Supported by DK43785).
• MUCOSAL IMMUNOSUPPRESSION BY INDUCTION OF APOPTOSIS IN LAMINA PROPRIA L Y M P t t O C Y T E S G.C. O~Sullivan, J.K. Collins, D. Collins, A. O'Mahony, and F. Shanahan. University College Cork, IRELAND. Therapeutic exploitation of a mechanism for selective suppression of activat©d lymphoeytes is desireable in several disorders. We previously desoribed and partially purified a factor produced by human esophageal squamous cell carcinomas that induces lmmunosuppression in vivo and in vitro (J Immunol 1993; 151:4847-56; Gastroenterology 1994; 106: A424). The tumor-derived factor induces apoptosis in peripheral blood lymphoeytes (PBL) but only when they are preactivated. Since normal intestinal lamina opria lymphoeytes (LPL) represent an activated immune compartment, we ve determined the activity of the tumor-derived immune suppressor factor (ISF) on human and marine LPL and examined its mechanism of action. The ISF was found to exert profound, dose-dependent, inhibition of resting and PHA-stimulated LPL proliferation (thymidine incorporation). When background apoptosis was controlled for (which was significantly greater in LPL than PBL from the same patient), the ISF induced significanl apoptosis as shown by morphology, flow cytometry, (propidium iodide staining with gating on eytokeratin-negative coils), and DNA gel eleetrophoresis. Because surface apo-1/FAS is an endogenous trigger mechanism of physiologic apoptosis, we determined whether ISF works through this pathway. To do this, we used PBL and LPL from MRL Ipr/lpr mice, which have a deficiency of functional apo-1/FAS receptor, and compared results with the MRL congenic control. The ISF was acuve on both FAS-mutant and FAS-normal lymphoeytes, indicating that it does not require an intact FAS signal pathway for apeptosis. To further clarify its mechanism of action, we measured the intraceUular cyclic AMP (cAMP) response to ISF in resting and activated lymphocytas, cAMP is negative regulator of activation and proliferation in lymphocytes. ISF induced significant elevation of cAMP levels to greater than 200pg/106 cells in activated lymphocytes, but resting lymphocytes did not respond to ISF with cAMP levels remaining at less than 20pg/106 cells, whereas both resting and activated cells responded to forskolin. In conclusion, the ISF of esophageal squamous cancer suppresses lymphocyte proliferation and induces apoptosis by increasing intracellular cAMP. The factor works through a pathway other than apo-I/FAS that appears to be expressed only in activated lymphocytes. Because LPL exist in a state of relative activation, they are exquisitely sensitive to this agent
which may ultimatelyhave therapeuticpotential.
• TNBS-INDUCED COLITIS IN RATS CAN BE TRANSFERRED BY T CELLS. MJHJ Palmen ~, OLC Wijburg ~, AS Pefiaz, SGM Meuwissen2, EP van Rees ~. Dept. of Cell Biology/Immunology ~ and Dept. of Gastroenterology 2, Faculty of Medicine, Vrije Universiteit, Amsterdam, The Netherlands. Introduction: Colitis can be induced in rats by intracolonic administration of 2,4,6-trinitrobenzene sulphonic acid (TNBS) in ethanol. As second administration of TNBS (subcutaneously) results in a prolonged chronic phase of inflammation, the rats probably have been sensitized by the first dose of TNBS, given in the colon (Palmen et al, Clin Exp Immunol 1995, in press). This suggests that T cells play an important role in the pathogenesis of experimental colitis. Therefore, the aim of the present study was to examine if T cell transfer from TNBS/ethanol sensitized rats induces colitis in naive recipients. Methods: Colitis was induced by a single rectal administration of 30 mg TNBS in ethanol 30%. Seventeen days later, T cells were isolated from the spleens and a high dose was administered intravenously to naive rats. As controls we administered T ceils of normal healthy rats and splenic leukocytes without T ceils to naive recipients. After 2 hours, either TNBS or saline was given intraperitoneally to the recipients. Three days later, the animals were sacrificed, macroscopic damage scores of the colon were established and macrophages, granulocytes and lymphocytes were studied in situ in the colon and small intestine using immunohistochemistry. Quantification was done using the IBAS image analysis system. Results: Edema was found in the colon of the recipients of T-cells obtained from TNBStreated rats, but not in the controls. An influx of granulocytes and macrophages was observed only in the colonic submucosa of recipients of sensitized T cells, which suggests that the animals suffer from an acute inflammation in the colon. We also observed an increase in the number of T cells in the colon. No effect was found in control rats. To examine which subset of T cells caused this effect, CD4+ T ceils were transferred, but this did not result in macroscopic or microscopic signs of inflammation. Conclusion: Colitis can be transferred to naive rats by T cells, obtained from rats with TNBS-ethanol induced experimental colitis. It might be possible that T cells recognize, CD8 involved, a specific protein in the colon in combination with TNBS.
Immunology, Microbiology, and Inflammatory Disorders A889
BENEFICIAL EFFECT OF APOCYNIN, A PLANT-DERIVED NADPH OXIDASE ANTAGONIST, IN TNBS-INDUCED EXPERIMENTAL COLITIS. MJHJ Palmen ~, CJ Beukelman3, RGM Mooij ~, SGM Meuwissen2, EP van ReesI. ~Dept of Cell Biology/Immunology, 2Dept of Gastroenterology, Fac. of Medicine, Vrije Universiteit, Amsterdam; ~Dept. of Pharmacognosy, Fac. Pharmacy, Universiteit Utrecht, The Netherlands. Introduction: Reactive oxygen species (ROS) produced by activated neutrophils and macrophages may play an important role in tissue damage in inflammatory bowel disease (IBD), The plantphenol 4-hydroxy-3-meth0xyacetophenone (trivial name: apocynin), an NADPH oxidase inhibitor, is newly isolated from traditional anti-inflammatory medicinal herb Picrorhiza kurroa. In the present study, the effect of apocynin in a rat model for IBD was studied. Methods: Colitis was induced in Wistar rats on day 0 by a single rectal administration of 30 mg TNBS in ethanol 30%. Rats were treated on day 0 and day 3 with 4 mg/kg apocynin i.v. Controls received saline i.v. in equal volumes. Rats were sacrificed on day 7 and macroscopical damage scores of the colon were determined on a 0-6 scale. Macrophages and granulocytes in the colon were studied in situ by immunohistochemistry and were quantified using the IBAS image analysis system. Furthermore, MPO activity was determined in colonic tissue, Results: Mean damage scores of the colon were significantly improved in the apocynin treated rats compared to the controls (mean damage score controls 3.4, mean damage scores after apocynin treatment 1). MPO activity was decreased with 98% in apocynin treated rats. Strikingly, a decrease was found in the number of granulocytes (67%) and macrophages (68%) in the colon. Conclusion: Apocynin has a striking beneficial effect on experimental colitis. The results suggest that treatment with apocynin, in addition to inhibiting ROS production, also decreases the influx of monocytes and neutrophils into the tissue.
ICAM-1 EXPRESSION IN THE SPLANCHNIC ORGANS IN VIVO~ EFFECTS OF ENDOTOXIN. J Pands, MA Perry#, A. Manning*, JM Russell, DC Anderson*, DN Granger. Dept Physiology. LSU Medical Center, Shreveport, LA 71111.
#Universitty of Soutn Wales, Sidney, Australia. *Upjohn Laboratories, Kalamazoo, M149001. ICAM-1 has been implicated in the recruitment of leukocytes into inflamed regions of the GI tract. However, there is no quantitative data in the literature that addressed the in vivo responses of this endothelial cell adhesion molecule to an inflammatory stimulus. The objectives of this study were to characterize and compare the constitutive and inducible expression of ICAM- 1 in splanchnic organs after exposure to endotoxin. Methods: ICAM- 1 expression was studied in the splanchnic organs of untreated or endotoxin-treated rats, at 3, 5, 9, 12, and 24 hours after endotoxin administration (5 mg/Kg, i.p.). ICAM-1 expression was measured using 125Ilabeled anti,rat ICAM-1 mAb (1A29), and an isotype matched control mAb: ~3~IP-23 (anti-dog ICAM-1) to correct for nonspecific accumulation of 1A29.ICAM-1 corrected activity was expressed as (% injected activity ~2sI-ICAM-1/g)- (% injected activity lzXI-P23/g). ICAM- 1 mRNA was measured in the intestine by Northern blot analysis. Results: ICAM-1 mAb binding was observed in all splanchnic organs. ~zsI-ICAM-1 mAb accumulation was blocked dose-dependently by increasing doses of cold ICAM- 1 mAb, whereas a non-binding mAb had no effect. The accumulation of ICAM-1 mAb varied widely in the different organs. It was high in the liver (4.4_-L-0.7%/g), spleen (4.2_+0.4%/g) and kidneys (1.3--+0.2 %/g), and much lower in stomach (0.06_+0.006 %/g) small bowel (0.15!-0.02) large bowel (0.14_+0:02 %/g), pancreas (0.09-20.01%/g) and mesentery (0.11_+0.01). Endotoxin induced an increase in ICAM- 1 mAb binding in the organs with a low basal expression: stomach (4-fold), and small intestine, large intestine, pancreas, and mesentery (3-fold), with no changes observed in liver, and. Maximal expression occurred at 9-12 hours after endotoxin administration, and persisted at 24 h. ICAM-1 mRNA was not detected in the small bowel under basal conditions, but a marked increase occurred at 3 h, i.e., preceding the significant increase in ICAM-1 expression at 5 h; Conclusions: ICAM-1 is constitutively expressed in all splanchnic organs in the rat_ The endothefial cell adhesion molecule is upregulated by endotoxin in those tissues with a low constitutive expression. Maximal upregulation occurs at 9-12 hours after the inflammatory stimulus. (Supported by DK43785).