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Immune regulation by intestinal smooth muscle cells (ISMCs)
A836
AGA ABSTRACTS
• THE CYTOSKELETON OF INTESTINAL EPITHELIAL CELLS PHYSICALLY CONSTRAINS THE TRANSEPITHELIAL MIGRATION OF NEUTROPHILS (PMNs) . . . . . P. Hofman. L. D'Andrea, S.P. Colgan, and J,L. 1V[adara. Division of Gastrointestinal Pathology, Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston MA. Transepithelial migration of PMN is a critical step in .many active inflammatory diseases of the lung, kidney, and intestine. It has recently been established that epithelial cells can release soluble factors which influence this transmigratory event. However, during PMN transmigration the epithelial cortical cytoskeleton is reordered raising the possibility that the epithelial cell may influence such PMN transit by direct'physical means as well. The polarized human intestinal crypt-like cell line, T84, was used to grOW biophysically confluent monolayers on permeable supports. PMNs were isolated from human blood and driven across epithelial mOnolayers by6 constructing transepithelial gradients of the chemotaetie peptide fMLP Off M). The dynamic rearrangemerit of the F-ectin cytoskeleton of epithelial cells is prevented by pro-loading with phalloidin while the normal regulation of a variety of membrane pumps and channels is maintained (J. Clin. Invest.87: 1903,1991 ). Epithelial microtubnles were depolymerized with noeodazole, r Monolayers were throughly washed following loading/treatment to prevent carryover of drugs to the eytoskeletal PMNs. PMN transmigration was qnantitated by a myeloperoxidase assay and epithelial barrier function was measured via standard biophysical techniques (resistance). Neither nocodazole or phalloidin substantially influenced baseline monolayer resistances (1200 vs 1500 vs 1250 ohm/era2 for nOcodazole vs phalloidin vs control). Confocal microscopy using appropriate labelling revealed a transmigration-associated rearrangement of epithelial~basolaterale actin which was attenuated by phaildidin loading and also eonf'n'med depolymerization of microtubules post nocodazole. Transepithelial migration of PMN was enhanced in phalloidinl loaded monolayers in comparison with controls but was unaffected in nocodazole treated monolayers (3.6x104:1:0.24 vs 1.104+ 0.42 vs 1.2 1045:0.2 respectively, p<.01) for phalloidin loading vs controls). The enhanced PMN transmigration across monolayers pre-loaded with phalloidin was also reflected in a greater transmigration-associated fall in resistance as compared with controls (3505:32 vs 480 + 43 ohm/era2 respectively, p<.02). Dynamic rearrangements of the epithelial f-actin cytoskeleton which occur during PMN transmigration may serve to constrain the rate in which PMN transmigrate. Thus epithelia may regulate, by active physical means, the transepithelial passage of PMN.
• IMMUNE REGULATION BY INTESTINAL SMOOTH MUSCLE CELLS (ISMCs). CM Hogaboam, CL Main, DP Snider, SM Collins. Intestinal Disease Research Programme and Vaccine Development Group, McMaster University, Hamilton, ON. Canada. Background~Aims: There is growing evidence that intestinal muscle cells are active participants, rather than innocent bystanders, in g u t inflammation, During intestinal inflammation, the muscularis exteraa is exposed to circulating antigens, and is a site of production of cytokines, such as interlenkin-lB, and the expression of MHC II and the intercellular adhesion molecule- 1 (ICAM- 1). These putative attributes of smooth muscle have been confirmed in vitro ns~ng muscle cells isolated from rodent small intestine. Taken together, these findings raise the possibility that muscle cells may engage and activate T lymphocytes through MHC H linked antigen presentation~ Methods: ISMCs were cultured from routine small ' intestine and immunocytochemicallystained for smooth muscle specific ~actin, MAC-l, MHC II and ICAM-1 prior to, and following, e×posure to IFN-'v for 48-96 h. Antigen presentation was investigated by testing the proliferation of T lymphocytes purified from mesenteric lymph nodes of mice sensitized to ovalbumin. ISMCs were exposed to IFN--r (!0-1000 U/mL) and ovalbumin (200 ~g/mL) for 48-96 h, washed and IL-2 treated T lymphocytes were added. T cell proliferation was assessed by [~H]thymidine incorporation 72 h later. Results: As we previously described, cultured ISMCs expressed smooth muscle specific a~actin (with or without IFN-3, treatment). No MAC-l-positive cells were identified in these cultures. Following stimulation with 1000 U/mL of IFN-~, for 72 h, muscle cells expressed MHC II and tCAM-I. In functional studies, untreated ISMCs did not induce T cell proliferation but when muscle cells were pretreated with 1000 U/mL IFN-~, and ovalbumin for a minimum of 72 h, significant T lymphocyte proliferation was detected. Exogenous IL-2 was #equired for this response and T cell proliferation was completely inhibited by anti-MHC II antibody. Conclusions: Smooth muscle cells take up, process and present antigen via MHC II and subsquently activate T lymphocytes. Thus, intestinal muscle cells possess the ability to modulate immune function in the gut. Supported by MRC Canada.
GASTROENTEROLOGY,Vol. 108, No. 4
• AN ORALLY ACTIVE NON-PEPTIDE ENDOTHELIN RECEPTOR ANTAGONIST (BOSENTAN)MARKEDLY REDUCES INJURY IN A RAT MODEL OF COLITIS. CM H0gaboam , MJ Muller, RH Hunt, SM Collins. Intestinal Disease Research Programme, McMaster University, Hamilton, ON. Canada. Background~Aims: Activation of endothelial cells by vasoactive mediators such as endothelin may be an early and strategically important step in the initiation Of inflammation in the intestine. Indeed, in view of recent evidence that inflammatory bowel disease is associated with upregulated expression of endothelin receptors on vascular endothelium (Gastroenterology 106-A2583, 1994), endothelin may be a potential therapeutic target i n IBD. The recent availability of an orally active endothelin receptor antagonist (bosentan;JPET 270:228-235,1994) allowed us to examine the role of endothelins in a rat model of colitis. Methods: Colitis Was inductd by intrarectal adminstrati0n Of trimtrobenzenesulfonic acid (TNB). In the treatment groups (n= 10/group), rats 'were gavaged with bosentan (10-100 mg/kg) 24 and 2 h prior to, or I h after, the induction" of colitis. On day 6 post-TNB, colonic tissues were removed for determination of macroscopic and microscopic tissue injury, and myeloperoxidase (MPO)activity. Results: TNB-induced colitis was typified by ulceration in the distal colon and a 35-fold increase in MPO activity compared to non-inflamed controls. When the treatment was given prior to the induction of colitis, daily, administration of bosentan dose~ dependently reduced colonic damage and MPO activity. Damage scores in rats treated with 100 mg/kg bosentan were reduced by 50%, although no inhibitory effect on colonic MPO activity was observed. However, the greatest beneficial effect of bosentan was observed at 30 mg/kg when colonic damage and granulocyte infiltration were attenuated by > 75 %. No bene:ficial effect o f bosentan a t any dose was seen when t h e pretreatment dosing regimen was excluded. Conclusions: These findings demonstrate that an orally active endothelin receptor antagonist can significantly reduce injury in an animal model of colitis and thus identify a novel therapeutic approach to the treatment of inflammatory bowel diseases. Bosentan was kindly provided by Hoffmann-LaRoche.
INTERLEUKIN-113 (II.,-1B) AND TUMOR NECROSIS FACTOR ALPHA (TNF~) POTENTIATE IMMUNE ACTIVATION BY INTESTINAL SMOOTH MUSCLE CELLS (ISMCs). CM. Hogaboam, DP Snider, SM Collins. Intestinal Disease Research Programme and Vaccine Development Group, McMaster University, Hamilton, ON. Background~Aims: We have shown that IL-1B and TNF-t~ are present in the muscularis externa during intestinal inflammation/Gastroenterology 107:691700,1994). In view of evidence that intestinal smooth muscle ceils directly participate in T cell activation, we examined whether IL-1B and TNF-ct potentiate the immunoregulatory properties of ISMCs. Methods: We have previously shown that INFw (1000 U/mL) induces MHC II on ISMCs at 72 h, but in the presense of IL-1B or TNF-a four to ten fold less IFN-3, was requited for comparable MHC II expression (Gastroenterology, 106:A1983, 1994). Neither IL~IB nor TNF9~, alone or in combination, induced MHC II expression on ISMCs. The functional capacity of cultured murine ISMCs to present antigen was investigated in a proliferation assay using T lymphocytes purified from mesenteric lymph nodes of mice sensitized to ovalbumin. ISMCs were exposed to IFN--y (100-1000 U/mL) and IL-1B (1-100 ng/mL) or TNFtx (0.5-5 ng/mL), together with ovalbumin for 72 h. The ISMCs were then washed and interleukin-2 (IL~2; 40 U/mL)-treated T lympl~ocyte preparations were added. T cell proliferation was assessed by [3H]thymidine incorporation 72 h later. Results: Untreated ISMCs did not induce T cell proliferation and neither IL-1B nor TNF~ alone caused T cell .proliferation. When muscle cells were pretreated with 1000 LVmLIFN-"/and ovalbumin for 72 h, significant T lymphocyte proliferation was detected. Although T cell proliferation was not induced by ISMCs previously exposed to 100 or 250 U/mL IFN-7, exposure of ISMCs to 100 ng/ml IL-11~and 100 U/mL IFN-), for 72 h resulted in a 3-fold increase in T cell proliferation. Stimulation of ISMCs with 5 ng/mL TNFt~ and 250 U/mL IFN-7 for the same time period induced a 6-fold increase in T cell proliferation. Conclusions: As demonstrated by T cell proliferation, IL-lg and TNFc~, two cytokines present in the external muscle layers during intestinal inflammation, potentiate the effects of' IFN-3, on ISMCs Thus. these findings provide further evidence that intestinal muscle ceils not only possess the ability to modulate immune function in the gut, but are in a cytokine-euriched environment w h i c h potentiates their ability to participate in immunoregulation during intestinal inflammation. Supported by MRC Canada.
AGA ABSTRACTS
• THE CYTOSKELETON OF INTESTINAL EPITHELIAL CELLS PHYSICALLY CONSTRAINS THE TRANSEPITHELIAL MIGRATION OF NEUTROPHILS (PMNs) . . . . . P. Hofman. L. D'Andrea, S.P. Colgan, and J,L. 1V[adara. Division of Gastrointestinal Pathology, Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston MA. Transepithelial migration of PMN is a critical step in .many active inflammatory diseases of the lung, kidney, and intestine. It has recently been established that epithelial cells can release soluble factors which influence this transmigratory event. However, during PMN transmigration the epithelial cortical cytoskeleton is reordered raising the possibility that the epithelial cell may influence such PMN transit by direct'physical means as well. The polarized human intestinal crypt-like cell line, T84, was used to grOW biophysically confluent monolayers on permeable supports. PMNs were isolated from human blood and driven across epithelial mOnolayers by6 constructing transepithelial gradients of the chemotaetie peptide fMLP Off M). The dynamic rearrangemerit of the F-ectin cytoskeleton of epithelial cells is prevented by pro-loading with phalloidin while the normal regulation of a variety of membrane pumps and channels is maintained (J. Clin. Invest.87: 1903,1991 ). Epithelial microtubnles were depolymerized with noeodazole, r Monolayers were throughly washed following loading/treatment to prevent carryover of drugs to the eytoskeletal PMNs. PMN transmigration was qnantitated by a myeloperoxidase assay and epithelial barrier function was measured via standard biophysical techniques (resistance). Neither nocodazole or phalloidin substantially influenced baseline monolayer resistances (1200 vs 1500 vs 1250 ohm/era2 for nOcodazole vs phalloidin vs control). Confocal microscopy using appropriate labelling revealed a transmigration-associated rearrangement of epithelial~basolaterale actin which was attenuated by phaildidin loading and also eonf'n'med depolymerization of microtubules post nocodazole. Transepithelial migration of PMN was enhanced in phalloidinl loaded monolayers in comparison with controls but was unaffected in nocodazole treated monolayers (3.6x104:1:0.24 vs 1.104+ 0.42 vs 1.2 1045:0.2 respectively, p<.01) for phalloidin loading vs controls). The enhanced PMN transmigration across monolayers pre-loaded with phalloidin was also reflected in a greater transmigration-associated fall in resistance as compared with controls (3505:32 vs 480 + 43 ohm/era2 respectively, p<.02). Dynamic rearrangements of the epithelial f-actin cytoskeleton which occur during PMN transmigration may serve to constrain the rate in which PMN transmigrate. Thus epithelia may regulate, by active physical means, the transepithelial passage of PMN.
• IMMUNE REGULATION BY INTESTINAL SMOOTH MUSCLE CELLS (ISMCs). CM Hogaboam, CL Main, DP Snider, SM Collins. Intestinal Disease Research Programme and Vaccine Development Group, McMaster University, Hamilton, ON. Canada. Background~Aims: There is growing evidence that intestinal muscle cells are active participants, rather than innocent bystanders, in g u t inflammation, During intestinal inflammation, the muscularis exteraa is exposed to circulating antigens, and is a site of production of cytokines, such as interlenkin-lB, and the expression of MHC II and the intercellular adhesion molecule- 1 (ICAM- 1). These putative attributes of smooth muscle have been confirmed in vitro ns~ng muscle cells isolated from rodent small intestine. Taken together, these findings raise the possibility that muscle cells may engage and activate T lymphocytes through MHC H linked antigen presentation~ Methods: ISMCs were cultured from routine small ' intestine and immunocytochemicallystained for smooth muscle specific ~actin, MAC-l, MHC II and ICAM-1 prior to, and following, e×posure to IFN-'v for 48-96 h. Antigen presentation was investigated by testing the proliferation of T lymphocytes purified from mesenteric lymph nodes of mice sensitized to ovalbumin. ISMCs were exposed to IFN--r (!0-1000 U/mL) and ovalbumin (200 ~g/mL) for 48-96 h, washed and IL-2 treated T lymphocytes were added. T cell proliferation was assessed by [~H]thymidine incorporation 72 h later. Results: As we previously described, cultured ISMCs expressed smooth muscle specific a~actin (with or without IFN-3, treatment). No MAC-l-positive cells were identified in these cultures. Following stimulation with 1000 U/mL of IFN-~, for 72 h, muscle cells expressed MHC II and tCAM-I. In functional studies, untreated ISMCs did not induce T cell proliferation but when muscle cells were pretreated with 1000 U/mL IFN-~, and ovalbumin for a minimum of 72 h, significant T lymphocyte proliferation was detected. Exogenous IL-2 was #equired for this response and T cell proliferation was completely inhibited by anti-MHC II antibody. Conclusions: Smooth muscle cells take up, process and present antigen via MHC II and subsquently activate T lymphocytes. Thus, intestinal muscle cells possess the ability to modulate immune function in the gut. Supported by MRC Canada.
GASTROENTEROLOGY,Vol. 108, No. 4
• AN ORALLY ACTIVE NON-PEPTIDE ENDOTHELIN RECEPTOR ANTAGONIST (BOSENTAN)MARKEDLY REDUCES INJURY IN A RAT MODEL OF COLITIS. CM H0gaboam , MJ Muller, RH Hunt, SM Collins. Intestinal Disease Research Programme, McMaster University, Hamilton, ON. Canada. Background~Aims: Activation of endothelial cells by vasoactive mediators such as endothelin may be an early and strategically important step in the initiation Of inflammation in the intestine. Indeed, in view of recent evidence that inflammatory bowel disease is associated with upregulated expression of endothelin receptors on vascular endothelium (Gastroenterology 106-A2583, 1994), endothelin may be a potential therapeutic target i n IBD. The recent availability of an orally active endothelin receptor antagonist (bosentan;JPET 270:228-235,1994) allowed us to examine the role of endothelins in a rat model of colitis. Methods: Colitis Was inductd by intrarectal adminstrati0n Of trimtrobenzenesulfonic acid (TNB). In the treatment groups (n= 10/group), rats 'were gavaged with bosentan (10-100 mg/kg) 24 and 2 h prior to, or I h after, the induction" of colitis. On day 6 post-TNB, colonic tissues were removed for determination of macroscopic and microscopic tissue injury, and myeloperoxidase (MPO)activity. Results: TNB-induced colitis was typified by ulceration in the distal colon and a 35-fold increase in MPO activity compared to non-inflamed controls. When the treatment was given prior to the induction of colitis, daily, administration of bosentan dose~ dependently reduced colonic damage and MPO activity. Damage scores in rats treated with 100 mg/kg bosentan were reduced by 50%, although no inhibitory effect on colonic MPO activity was observed. However, the greatest beneficial effect of bosentan was observed at 30 mg/kg when colonic damage and granulocyte infiltration were attenuated by > 75 %. No bene:ficial effect o f bosentan a t any dose was seen when t h e pretreatment dosing regimen was excluded. Conclusions: These findings demonstrate that an orally active endothelin receptor antagonist can significantly reduce injury in an animal model of colitis and thus identify a novel therapeutic approach to the treatment of inflammatory bowel diseases. Bosentan was kindly provided by Hoffmann-LaRoche.
INTERLEUKIN-113 (II.,-1B) AND TUMOR NECROSIS FACTOR ALPHA (TNF~) POTENTIATE IMMUNE ACTIVATION BY INTESTINAL SMOOTH MUSCLE CELLS (ISMCs). CM. Hogaboam, DP Snider, SM Collins. Intestinal Disease Research Programme and Vaccine Development Group, McMaster University, Hamilton, ON. Background~Aims: We have shown that IL-1B and TNF-t~ are present in the muscularis externa during intestinal inflammation/Gastroenterology 107:691700,1994). In view of evidence that intestinal smooth muscle ceils directly participate in T cell activation, we examined whether IL-1B and TNF-ct potentiate the immunoregulatory properties of ISMCs. Methods: We have previously shown that INFw (1000 U/mL) induces MHC II on ISMCs at 72 h, but in the presense of IL-1B or TNF-a four to ten fold less IFN-3, was requited for comparable MHC II expression (Gastroenterology, 106:A1983, 1994). Neither IL~IB nor TNF9~, alone or in combination, induced MHC II expression on ISMCs. The functional capacity of cultured murine ISMCs to present antigen was investigated in a proliferation assay using T lymphocytes purified from mesenteric lymph nodes of mice sensitized to ovalbumin. ISMCs were exposed to IFN--y (100-1000 U/mL) and IL-1B (1-100 ng/mL) or TNFtx (0.5-5 ng/mL), together with ovalbumin for 72 h. The ISMCs were then washed and interleukin-2 (IL~2; 40 U/mL)-treated T lympl~ocyte preparations were added. T cell proliferation was assessed by [3H]thymidine incorporation 72 h later. Results: Untreated ISMCs did not induce T cell proliferation and neither IL-1B nor TNF~ alone caused T cell .proliferation. When muscle cells were pretreated with 1000 LVmLIFN-"/and ovalbumin for 72 h, significant T lymphocyte proliferation was detected. Although T cell proliferation was not induced by ISMCs previously exposed to 100 or 250 U/mL IFN-7, exposure of ISMCs to 100 ng/ml IL-11~and 100 U/mL IFN-), for 72 h resulted in a 3-fold increase in T cell proliferation. Stimulation of ISMCs with 5 ng/mL TNFt~ and 250 U/mL IFN-7 for the same time period induced a 6-fold increase in T cell proliferation. Conclusions: As demonstrated by T cell proliferation, IL-lg and TNFc~, two cytokines present in the external muscle layers during intestinal inflammation, potentiate the effects of' IFN-3, on ISMCs Thus. these findings provide further evidence that intestinal muscle ceils not only possess the ability to modulate immune function in the gut, but are in a cytokine-euriched environment w h i c h potentiates their ability to participate in immunoregulation during intestinal inflammation. Supported by MRC Canada.