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The role of macrophages in experimental colitis is dependent upon the manner in which the inflammation is induced
AGAA573
April 2000
2988
2990
LEUKOCYTE HOMING IN DSS-INDUCED COLITIS IS REDUCED BY MADCAM-l BLOCKADE IN VIVO. Stefan A. Farkas, Hans Herfarth, Matthias Roessle, Karl-Walter Jauch, Matthias Anthuber, Univ of Regensburg, Dept of Surg, Regensburg, Germany; Univ of Regensburg, Dept of Internal Medicine I, Regensburg, Germany. Background: Leukocyte adherence and further extravasation plays a pivotal role in the pathogenesis of chronic inflammatory bowel (IBD) disease. Recent results support that MadCAM-l is exclusively expressed in the intestine and associated lymphoid tissue and participates in intestinal leukoctye homing. Therefore, we investigated the expression of MadCAM-I and, by means of in vivo microscopy, we analyzed the effect of MadCAM-l blockade on leukocyte homing in chronic DSS-induced colitis. Methods: Chronic colitis was induced by oral administration of 4 cycles of 5% DSS dissolved in the drinking water given for 7 days and 10 days of normal water to Balb/c mice. 14 days after the last cycle, treatment with anti-MadCAM-l or isotyp control antibody was administered by ip administration 3 times over 7 days (n=5/group). In vivo microscopy: In anaesthetized mice colon microcolonic circulatory networks and leukocytes were visualized by i.v. injection of Fl'I'Cvdextran and acridin orange respectively. Permanent adherent leukocytes (sticker) were assessed in ten randomly selected collecting venules (CV) and postcapillary venules (PV). Finally, the colon was incised on the antimesenterial side, the colonic mucosa was visualized and extravasated leukocytes were counted. Results: MadCAM-l expression, revealed by imrnunhistochemistry, was upregulated in the endothelium of mice with chronic colitis. DSS administration resulted in a increase of sticker in PV and CV (p
THE ROLE OF MACROPHAGES IN EXPERIMENTAL COLITIS IS DEPENDENT UPON THE MANNER IN WHICH THE INFLAMMATION IS INDUCED. David C. Ford, Francesca Galeazzi, Patricia A. Blennerhassett, Robert H. Riddell, Stephen M. Collins, McMaster Univ, Hamilton, ON, Canada; Univ of Padova, Padova, Italy. Macrophages play a critical role in defense of the gut by virtue of their ability to present antigen. Macrophages are also a source of proinflammatory cytokines that induce tissue injury and are thus a target for treatment. The extent to which protective and injurious properties of macrophages contribute to the expression of IBD is incompletely understood. To evaluate this, we examined the role of macrophages in two models of experimental colitis by performing studies in op/op mice that are deficient in macrophage-colony stimulating factor (M-CSF) and therefore incapable of macrophage maturation and activation. Colitis was induced in op/op and M-CSF expressing opt? mice by (1) adding 6% Dextran Sodium Sulfate (DSS) to the drinking water or (2) by administering 3mg dinitrobenzenesulfonic acid (DNB)in 50% ethanol intra-rectally. Histological damage scores and myeloperoxidase activity (MPO) were used as objective measures. Both DNB and DSS induced an acute colitis with microscopical damage with ulceration evident in both models as well as >20-fold increase in MPO activity within 3-5 days. Colitis induced by DNB was substantially more severe in op/op compared to opt? mice, with a >50% mortality rate and a two-fold increase in macroscopical and microscopical damage and MPO increase particularly by 24h. While there was a large increase in F4/80+ve macrophages in opt? mice with colitis, this was markedly attenuated in op/op mice. In contrast, acute colitis induced by DSS was not more severe in op/op mice compared to their M-CSF-bearing littermates. Weight loss and diarrhea were similar in op/op and opt? mice (wt loss 8.3% in op/op v 6.6% in opt? P>0.05; diarrhea score 2.5 v 2.3 in op/op and opt? respectively, P>0.05). MPO activity was 2.7±2.l and 2.6±0.9 U/mg in op/op and opt? mice respectively (P>0.05). Indeed, there was an improved histological score in the op/op mice compared to opt? mice (9.6±3.2 v 13.4±3.5, respectively P=0.024). While few F4/80+ve cells were evident in op/op without colitis, an increase in these cells following colitis was only evident in opt? mice. These results indicate that the absence of an M-CSF-dependent macrophage response is deleterious and fatal in DNB-induced colitis, but not in DSS colitis where its role appears minimal. We conclude that the role of macrophages in acute colitis is dependent on the nature of the inflammatory stimulus. This has implications for the use of these models in evaluating therapies directed against macrophages or their products. Supported by MRC Canada.
Results: Control ossnsotyp control OSS/antl MBdCAM·1
Sticker InPV leukocytes/mm2
StIcker inCV venular surface
Extravasation leukocyteslmm2 mucosa
8±3 l37±10 9O±1
2±1 34±1 27±5
19±4 52±8 21±3
2989
2991
GLIOTOXIN IS AN EFFECTIVE INHIBITOR OF IN VITRO AND IN VIVO EFFECTS INDUCED BY BACTERIAL PEPTIDOGLYCAN·POLYSACCHARIDE (PG-PS). Leo R. Fitzpatrick, Jian Wang, True V. Le, Maryland Research Lab, Rockville, MD. Previously, we found that gliotoxin (a fungal metabolite) inhibited cytokine production in vitro, as well as indices of colitis in rats (Fitzpatrick et al., Gastroenterology 116:A715, 1999). Aims: The aims of these studies were: 1) to investigate how gliotoxin inhibited PG-PS induced cytokine production, and 2) to further evaluate the efficacy of gliotoxin against PG-PS induced colitis in rats. Methods: In vitro experiments utilized a rat macrophage cell line (NR8383). PG-PS (100 J.Lg/ml) stimulated TNF-a production was measured by ELISA. Nuclear binding of NF-KBwas evaluated by EMSA. In vivo, colitis was induced by 9 injections of PG-PS to a 5 ern segment of distal rat colon. Gliotoxin (I mglkg) or saline vehicle were administered bid to Lewis rats by ip injection (n = 12 or 13 per group). Dosing was for one week, starting on day 14 after colitis induction. Various macroscopic and biochemical indices (MPO activity, IL-II3, TNF-a) were used to assess the colitis. Any associated arthritis was quantitated, by measuring the rear ankle joint diameters (in mm) of rats. Results: Gliotoxin inhibited PG-PS stimulated TNF-a production (ICso = 0.6 J.LM). This compound also inhibited the PG-PS induced nuclear binding of NF-KB (ICso = 1.8 J.LM). There was a good correlation (r = 0.986) between the percent inhibitions of these two parameters. Gliotoxin, at 2= 3 J.LM, fully inhibited the in vitro effects induced by PG-PS. On day 21 of PG-PS induced colitis, gliotoxin significantly attenuated (p < 0.05 vs. vehicle) macroscopic damage to the colon (i.e., the % incidence of colonic adhesions and nodules). In this regard, the mean colonic injury score (0 to 5 scale)was 4.5±0.2 in vehicle treated rats, but only 2.1:+:0.5 in gliotoxin treated rats. With gliotoxin treatment, there was some normalization (from 30 to 43%) of MPO,IL-II3, and TNF-a. The mean ankle joint diameter of gliotoxin treated rats was partially normalized (by 43%) compared to vehicle treated animals. Vehicle treated rats gained weight (12±3 grams), over the one-week dosing period. In contrast, gliotoxin treated animals tended to lose weight (-2±2 grams). Conclusions: Gliotoxin reduced PG-PS stimulated TNF-a production in macrophages by a NF-KB dependent mechanism, and also improved several indices of PG-PS induced colitis in rats. Gliotoxin also had an anti-arthritic effect in PG-PS treated animals. However, gliotoxin treated rats did show some evidence of mild toxicity. These results indicate that a NF-KB inhibitor can effectively treat colitis in rats.
DYSREGULATION OF INTERLEUKIN-7 MEDIATED IMMUNE RESPONSES IN CHRONIC INFLAMED MUCOSA OF T CELL RECEPTOR ALPHA CHAIN MUTANT MOUSE. Kazuto Fukui, Mamoru Watanabe, Tomoharu Yajima, Susumu Okamoto, Motomi Yamazaki, Hiromasa Ishii, Toshifumi Hibi, Keio Univ, Tokyo, Japan; Keio Cancer Ctr, Tokyo, Japan. Background: We have demonstrated interleukin-7 (IL-7) production by colonic epithelial cells and dysregulation of mucosal IL-7 mediated immune responses in the colitis lesion of human ulcerative colitis and IL-7 transgenic mice. To clarify the role of IL-7 in the development of colitis, we investigated mucosal IL-7 system in T-cell receptor alpha chain mutant (TCRa-I-) mice colitis model and assessed the therapeutic approaches targeting IL-7 signal pathway. Methods: We assessed histological findings, IL-7 expression in the epithelial cells and IL-7 receptor expression in mucosal lymphocytes in the colonic mucosa of TCRa-l- mice. To elucidate the role of mucosal IL-7/1L-7 receptor (IL-7R) signals in the development of chronic colitis, we established TCRa-l- X IL-7R-I- double knockout mice. Results: IL-7 expression in the colonic epithelial cells was normal at 4-6 weeks of age, but significantly decreased in the TCRa-l- mice at 11-16 weeks with development of chronic colitis. With decreased expression of IL-7, IL-7R positive T cells were infiltrated in the inflamed lamina propria of those mice. More than 95% of CD4+TCRl3dim cells that mediated inflammation expressed IL-7R in the chronic inflamed colonic mucosa. Recombinant IL-7 injection into TCR a-I- mice decreased IL-7R positive cells in the colonic lamina propria. TCRa-l- X IL-7R-I- double knockout never developed colitis. Moreover, IL-7 expression was not decreased in the epithelial cells and CD4+ TCRl3dimIL-7 receptor positive T cells were not infiltrated in the lamina propria of double knockout mice. Conclusion: Dysregulation of IL-7 may involve in the development of chronic colitis in TCR emutant mice. We are now under the investigation of new therapeutic approaches targeting IL-7/1L-7 signal pathway in the treatment of chronic colitis.
April 2000
2988
2990
LEUKOCYTE HOMING IN DSS-INDUCED COLITIS IS REDUCED BY MADCAM-l BLOCKADE IN VIVO. Stefan A. Farkas, Hans Herfarth, Matthias Roessle, Karl-Walter Jauch, Matthias Anthuber, Univ of Regensburg, Dept of Surg, Regensburg, Germany; Univ of Regensburg, Dept of Internal Medicine I, Regensburg, Germany. Background: Leukocyte adherence and further extravasation plays a pivotal role in the pathogenesis of chronic inflammatory bowel (IBD) disease. Recent results support that MadCAM-l is exclusively expressed in the intestine and associated lymphoid tissue and participates in intestinal leukoctye homing. Therefore, we investigated the expression of MadCAM-I and, by means of in vivo microscopy, we analyzed the effect of MadCAM-l blockade on leukocyte homing in chronic DSS-induced colitis. Methods: Chronic colitis was induced by oral administration of 4 cycles of 5% DSS dissolved in the drinking water given for 7 days and 10 days of normal water to Balb/c mice. 14 days after the last cycle, treatment with anti-MadCAM-l or isotyp control antibody was administered by ip administration 3 times over 7 days (n=5/group). In vivo microscopy: In anaesthetized mice colon microcolonic circulatory networks and leukocytes were visualized by i.v. injection of Fl'I'Cvdextran and acridin orange respectively. Permanent adherent leukocytes (sticker) were assessed in ten randomly selected collecting venules (CV) and postcapillary venules (PV). Finally, the colon was incised on the antimesenterial side, the colonic mucosa was visualized and extravasated leukocytes were counted. Results: MadCAM-l expression, revealed by imrnunhistochemistry, was upregulated in the endothelium of mice with chronic colitis. DSS administration resulted in a increase of sticker in PV and CV (p
THE ROLE OF MACROPHAGES IN EXPERIMENTAL COLITIS IS DEPENDENT UPON THE MANNER IN WHICH THE INFLAMMATION IS INDUCED. David C. Ford, Francesca Galeazzi, Patricia A. Blennerhassett, Robert H. Riddell, Stephen M. Collins, McMaster Univ, Hamilton, ON, Canada; Univ of Padova, Padova, Italy. Macrophages play a critical role in defense of the gut by virtue of their ability to present antigen. Macrophages are also a source of proinflammatory cytokines that induce tissue injury and are thus a target for treatment. The extent to which protective and injurious properties of macrophages contribute to the expression of IBD is incompletely understood. To evaluate this, we examined the role of macrophages in two models of experimental colitis by performing studies in op/op mice that are deficient in macrophage-colony stimulating factor (M-CSF) and therefore incapable of macrophage maturation and activation. Colitis was induced in op/op and M-CSF expressing opt? mice by (1) adding 6% Dextran Sodium Sulfate (DSS) to the drinking water or (2) by administering 3mg dinitrobenzenesulfonic acid (DNB)in 50% ethanol intra-rectally. Histological damage scores and myeloperoxidase activity (MPO) were used as objective measures. Both DNB and DSS induced an acute colitis with microscopical damage with ulceration evident in both models as well as >20-fold increase in MPO activity within 3-5 days. Colitis induced by DNB was substantially more severe in op/op compared to opt? mice, with a >50% mortality rate and a two-fold increase in macroscopical and microscopical damage and MPO increase particularly by 24h. While there was a large increase in F4/80+ve macrophages in opt? mice with colitis, this was markedly attenuated in op/op mice. In contrast, acute colitis induced by DSS was not more severe in op/op mice compared to their M-CSF-bearing littermates. Weight loss and diarrhea were similar in op/op and opt? mice (wt loss 8.3% in op/op v 6.6% in opt? P>0.05; diarrhea score 2.5 v 2.3 in op/op and opt? respectively, P>0.05). MPO activity was 2.7±2.l and 2.6±0.9 U/mg in op/op and opt? mice respectively (P>0.05). Indeed, there was an improved histological score in the op/op mice compared to opt? mice (9.6±3.2 v 13.4±3.5, respectively P=0.024). While few F4/80+ve cells were evident in op/op without colitis, an increase in these cells following colitis was only evident in opt? mice. These results indicate that the absence of an M-CSF-dependent macrophage response is deleterious and fatal in DNB-induced colitis, but not in DSS colitis where its role appears minimal. We conclude that the role of macrophages in acute colitis is dependent on the nature of the inflammatory stimulus. This has implications for the use of these models in evaluating therapies directed against macrophages or their products. Supported by MRC Canada.
Results: Control ossnsotyp control OSS/antl MBdCAM·1
Sticker InPV leukocytes/mm2
StIcker inCV venular surface
Extravasation leukocyteslmm2 mucosa
8±3 l37±10 9O±1
2±1 34±1 27±5
19±4 52±8 21±3
2989
2991
GLIOTOXIN IS AN EFFECTIVE INHIBITOR OF IN VITRO AND IN VIVO EFFECTS INDUCED BY BACTERIAL PEPTIDOGLYCAN·POLYSACCHARIDE (PG-PS). Leo R. Fitzpatrick, Jian Wang, True V. Le, Maryland Research Lab, Rockville, MD. Previously, we found that gliotoxin (a fungal metabolite) inhibited cytokine production in vitro, as well as indices of colitis in rats (Fitzpatrick et al., Gastroenterology 116:A715, 1999). Aims: The aims of these studies were: 1) to investigate how gliotoxin inhibited PG-PS induced cytokine production, and 2) to further evaluate the efficacy of gliotoxin against PG-PS induced colitis in rats. Methods: In vitro experiments utilized a rat macrophage cell line (NR8383). PG-PS (100 J.Lg/ml) stimulated TNF-a production was measured by ELISA. Nuclear binding of NF-KBwas evaluated by EMSA. In vivo, colitis was induced by 9 injections of PG-PS to a 5 ern segment of distal rat colon. Gliotoxin (I mglkg) or saline vehicle were administered bid to Lewis rats by ip injection (n = 12 or 13 per group). Dosing was for one week, starting on day 14 after colitis induction. Various macroscopic and biochemical indices (MPO activity, IL-II3, TNF-a) were used to assess the colitis. Any associated arthritis was quantitated, by measuring the rear ankle joint diameters (in mm) of rats. Results: Gliotoxin inhibited PG-PS stimulated TNF-a production (ICso = 0.6 J.LM). This compound also inhibited the PG-PS induced nuclear binding of NF-KB (ICso = 1.8 J.LM). There was a good correlation (r = 0.986) between the percent inhibitions of these two parameters. Gliotoxin, at 2= 3 J.LM, fully inhibited the in vitro effects induced by PG-PS. On day 21 of PG-PS induced colitis, gliotoxin significantly attenuated (p < 0.05 vs. vehicle) macroscopic damage to the colon (i.e., the % incidence of colonic adhesions and nodules). In this regard, the mean colonic injury score (0 to 5 scale)was 4.5±0.2 in vehicle treated rats, but only 2.1:+:0.5 in gliotoxin treated rats. With gliotoxin treatment, there was some normalization (from 30 to 43%) of MPO,IL-II3, and TNF-a. The mean ankle joint diameter of gliotoxin treated rats was partially normalized (by 43%) compared to vehicle treated animals. Vehicle treated rats gained weight (12±3 grams), over the one-week dosing period. In contrast, gliotoxin treated animals tended to lose weight (-2±2 grams). Conclusions: Gliotoxin reduced PG-PS stimulated TNF-a production in macrophages by a NF-KB dependent mechanism, and also improved several indices of PG-PS induced colitis in rats. Gliotoxin also had an anti-arthritic effect in PG-PS treated animals. However, gliotoxin treated rats did show some evidence of mild toxicity. These results indicate that a NF-KB inhibitor can effectively treat colitis in rats.
DYSREGULATION OF INTERLEUKIN-7 MEDIATED IMMUNE RESPONSES IN CHRONIC INFLAMED MUCOSA OF T CELL RECEPTOR ALPHA CHAIN MUTANT MOUSE. Kazuto Fukui, Mamoru Watanabe, Tomoharu Yajima, Susumu Okamoto, Motomi Yamazaki, Hiromasa Ishii, Toshifumi Hibi, Keio Univ, Tokyo, Japan; Keio Cancer Ctr, Tokyo, Japan. Background: We have demonstrated interleukin-7 (IL-7) production by colonic epithelial cells and dysregulation of mucosal IL-7 mediated immune responses in the colitis lesion of human ulcerative colitis and IL-7 transgenic mice. To clarify the role of IL-7 in the development of colitis, we investigated mucosal IL-7 system in T-cell receptor alpha chain mutant (TCRa-I-) mice colitis model and assessed the therapeutic approaches targeting IL-7 signal pathway. Methods: We assessed histological findings, IL-7 expression in the epithelial cells and IL-7 receptor expression in mucosal lymphocytes in the colonic mucosa of TCRa-l- mice. To elucidate the role of mucosal IL-7/1L-7 receptor (IL-7R) signals in the development of chronic colitis, we established TCRa-l- X IL-7R-I- double knockout mice. Results: IL-7 expression in the colonic epithelial cells was normal at 4-6 weeks of age, but significantly decreased in the TCRa-l- mice at 11-16 weeks with development of chronic colitis. With decreased expression of IL-7, IL-7R positive T cells were infiltrated in the inflamed lamina propria of those mice. More than 95% of CD4+TCRl3dim cells that mediated inflammation expressed IL-7R in the chronic inflamed colonic mucosa. Recombinant IL-7 injection into TCR a-I- mice decreased IL-7R positive cells in the colonic lamina propria. TCRa-l- X IL-7R-I- double knockout never developed colitis. Moreover, IL-7 expression was not decreased in the epithelial cells and CD4+ TCRl3dimIL-7 receptor positive T cells were not infiltrated in the lamina propria of double knockout mice. Conclusion: Dysregulation of IL-7 may involve in the development of chronic colitis in TCR emutant mice. We are now under the investigation of new therapeutic approaches targeting IL-7/1L-7 signal pathway in the treatment of chronic colitis.