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Increased oxidation & nitration injury in intestinal mucosa of patients with IBD as detected by a highly sensitive immunoblot
April 2000
spreading and tight junction remodeling response by neighboring viable cells.
4265 PEROXYNITRITE-INDUCED NITRATION & OXIDATION IN CYTOSKELETAL INSTABILITY & LOSS OF INTESTINAL EPITHELIAL BARRIER FUNCTION (BF). Ali Banan, Sandeep Choudhary, Yang Zhang, Ali Keshavarzian, Rush Univ, Chicago, IL. Peroxynitrite (ONOO, a potent oxidizing agent) has been detected in the inflamed mucosa of several GI disorders (e.g. inflammatory bowel disease, IBD). Recent reports have proposed that ONOO is responsible for injury & loss of intestinal BF in IBD, but the underlying mechanism remains unexplored. We recently showed the importance of microtubule (MT) cytoskeleton in the maintenance of intact intestinal BF (Banan JPET, 291: 1075-1085, 1999). For the current investigation we hypothesized that ONOO-induced loss of intestinal BF is caused by nitration, oxidation, & disassembly / disruption of MT cytoskeleton. Methods: We evaluated the effects of several ONOO donors on epithelial BF, oxidation & nitration of tubulin (structural backbone of MT) & cytoskeletal disassembly / instability (n = 6 per group & *p<0.05). Human colonic cell (Caco-2) monolayers were preincubated in authentic ONOO or ONOO donors [morpholinosydnoimine (SIN-I) or S-3-nitroso-N-acetyl penicillamine (SNAP) + Xanthine (X) / X Oxidase] in the range of 0.01 - 5 mM :!: selective antioxidants [L-Cysteine, SOD (superoxide dismutase) or the inactive analouges DCysteine & iSOD, respectively]. Immunoblolling of fractionated polymerized tubulin (S2; index of stability) & the monomeric tubulin (SI; index of disruption) to detect oxidation, nitration, & disassembly of tubulin as well as immunofluorescent staining of MT followed by detailed evaluation by high-resolution laser confocal microscopy were performed. BF was determined by fluorescein sulfonic acid clearance. Results: ONOO or donors dose-dependently & significantly [1] t the stable S2 polymerized tubulin, [2] t the unstable Sl tubulin s, in concert, [3] t two measures of oxidative stress (fluorescence of dichlorofluorescein) & oxidation & nitration of tubulin (i.e., foot prints of ONOO generation). These compounds also dose-dependently & significantly disrupted BF & extensively disassembled / damaged MT. These effects were completely prevented by pretreatment with selective anti-oxidant scavengers: L-Cysteine or SOD but not their inactive analouges D-Cysteine or iSOD. Conclusions: Data demonstrate, for the first time, that oxidation, nitration, & disassembly of the MT cytoskeleton induced by ONOO causes the disruption of intestinal epithelial barrier function. Thus, the instability of this critical component may play an essential role in abnormal intestinal barrier function in inflammatory GI disorders associated with the presence of ONOO.
AGAAS03
4267 PROINFLAMMATORY CYTOKINES DISTURB ELECTROGENIC SODIUM ABSORPTION: A MECHANISM OF COLONIC DYSFUNCTION IN AN IBD MODEL. Christian Barmeyer, Ingo Grotjohann, Heinz Schmitz, Peter Scholz, Joachim Mankertz, Ernst O. Riecken, Michael Fromm, Joerg D. Schulzke, Univ Hosp Benjamin Franklin, Berlin, Germany; Schering AG, Berlin, Germany. We have recently shown that diarrhea in ulcerative colitis (UC) is caused by an impaired barrier function and that this defect is mediated by TNFO' and IFNy. An other mechanism contributing to diarrhea is impaired Na+ absorption in the distal colon. We studied the effect of these two cytokines as well as of IL-I 13 on active electrogenic Na + absorption in rat late distal colon in vitro. Using the same tissues we analyzed the mRNA expression of the 3 subunits of the epithelial Na+ channel (ENaC). Tissues were incubated in the Ussing chamber for 6 hours with TNFO'alone (loo ng/ml), a high dose combination of TNFO'+ IFNy (100 ng/ml + 100 Vlml), a low dose combination ofTNFO'+IFNy (10 ng/ml + 10 U/ml), IFNy alone (100 U/ml), and IL-If3 alone (10 ng/ml). Then, 3 nM aldosterone was added and short circuit current (lsc) was monitored for additional 8 hours. Finally, electrogenic Na+ absorption (JNa in p.moleh-1ecm-2) was quantified by addition of 10-4 M amiloride. SUbse~uently, the 0'-, 13- and y-subunit mRNA expression of the epithelial Na channel (ENaC) was detected by Northern blot analysis. As shown in the Table, treatment with either TNFO', TNFO'+ IFNy (high conc.) or IL-lf3 reduced JN a if compared to control. In parallel, Northern blot analysis revealed a lower RNA expression of ENaC 13- and y-subunits, whereas the a-subunit was unchanged. Neither TNFO'+ IFNy (low conc.) nor IFNy alone altered I N • or RNA expression of any of the 3 ENaC subunits. In conclusion: Proinflammatory cytokines TNFO' and IL-If3lead to impaired electrogenic Na" absorption in rat late distal colon as well as to a down-regulation of mRNA expression of the ENaC 13- and y-subunits, while that of the a-subunit was unchanged. IFN-y can intensify the effect of TNFO'. This may represent a distinct pathomechanism in colonic inflammation contributing to diarrhea as e.g. in IBD.
TNFu+IFNy
J", % ofcontrol
n
Pv,lu,
control
high cone.
lowcone.
TNFu
IFNy
IL.lp
13.4±1.1 (100) 11
1.8±0.5 14 8 <0.001
11.7±2.3 88 13 n.s.
5.9±1.1 44 10 <0.001
16.5±2.4 123 10
3.5±08 26 10 <0.001
n.s.
4266 INCREASED OXIDATION & NITRATION INJURY IN INTESTINAL MUCOSA OF PATIENTS WITH IBD AS DETECTED BY A HIGHLY SENSITIVE IMMUNOBLOT. Yang Zhang, Ali Banan, Todd M. Murphy, Sandeep Choudhary, Ali Keshavarzian, Rush Univ, Chicago, IL. Nitric oxide (NO) overproduction & generation of peroxynitrite (ONOO, a potent oxidizer) has been proposed to be responsible for mucosal injury in IBD disorders. As a highly toxic reactive species, ONOO has been suggested to indiscriminately attack biomolecules essential to mucosal function. However, the critical contribution of ONOO to the pathogenesis of IBD remains unclear. Aims: To determine [I] the native levels of oxidation I nitration in the intestinal mucosal biopsies of normal subjects; [2] whether increased oxidation (carbonylation) & nitrotyrosination (nitration, a foot print of ONOO generation) can be found in mucosal biopsies of IBD patients. Methods: We have developed a highly reliable, rapid & sensitive quantitative immunblot that can detect oxidative alterations in flash frozen mucosal biopsies [n= 9 - 16 I group, p<0.05, (*) denotes significance] from control (CON) & patients with active (a) or inactive (i) Colitis [Ulcerative Colitis (UC), Crohn s (CC) & Specific Colitis (SC)] using monoclonal anti-carbonyl & anti-nitrotyrosine antibodies that were conjugated to peroxidase by 3 protein cross-linking with 0.2% gluteraldehyde. Two-dimensional (2-D) densitometry was then performed to quantitate oxidative changes. Results: Endoscopic colonic mucosa from CON exhibited a low level of native carbonyl (5.6 :!: 1.2%) & nitration (l9.2 ::': 1.9%) immunoreactivity. In contrast, colonic mucosa of patients with active colitis showed (l2 & 4 fold, respectively) increased (*) levels of both carbonyl [aUC, 71 :!: 5.8%; aCC, 87.5 ::': 11.8%; aSC, 91.4 :!: 6.4%] & nitration [75 ::': 12%; 87 :!: 10%; 48 ::': 1.2%, respectively]. When colitis was in remission levels of nitration [iUC, 50 :!: 1.3%; iCC, 65 :!: 10%] & carbonyl [iUC, 52.1 ::': 5.7%; iCC, 22.4 ::': 7.4%] decreased (*). Conclusions: Data demonstrate, for the first time, [1] that abnormally high immunoreactive levels of carbonyl & nitrotyrosine moieties ( foot prints of ONOO) are present in the inflamed colonic mucosa of patients with active colitis. Thus, oxidative damage may be an essential underlying cause of mucosal injury in Ulcerative Colitis & Crohn s Disease. However, these alterations are not specific to such disorders, as they are also present in Specific Colitis. [2] Mucosal oxidation / nitration is a useful marker of assessing the degree of oxidative stress & may be used as an index of disease activity in IBD.
4268 INCREASED TIGHT JUNCTIONAL PERMEABILITY ASSOCIATED WITH CROHN'S DISEASE AND ULCERATIVE COLITIS. Vikram Barpujari, James J. Thornton, James M. Mullin, Hilary M. Clarke, Alejandro Peralta Soler, The Lankenau Hosp, Wynnewood, PA; The Lankenau Med Research Ctr, Wynnewood, PA. Aim: The intestinal epithelium acts as a barrier between two fluid compartments. During an inflammatory process, the intestinal epithelium must preserve its barrier function to maintain tissue homeostasis. Previous studies have shown abnormal intestinal permeability in patients with Crohn's disease and ulcerative disease. The purpose of this study was to assess the permeability of tight junctions (TJ) of the intestinal epithelium of patients with Crohn' s disease and ulcerative colitis (UC), applying an in situ method that uses an electron-dense dye and electron microscopy. Methods: Colon pinch biopsies were placed in chambers designed specifically for this study, with the mucosa facing up. The epithelium was exposed for 30 min at room temperature to 2.5% glutaraldehyde in O.IM sodium cacodylate buffer pH 7.4 containing 0.3% ruthenium red (RR). After rinsing, the epithelium was exposed to 2% osmium tetroxide and 0.3% RR for 30 min. Samples were rinsed and processed for electron microscopy. Ultrathin sections were cut perpendicular to the surface in a Reichert Ultracut ultramicrotome and photographed in a JEOL 1200 electron microscope, for quantitative analysis of TJ permeability. Results: Analysis of 4 non-IBD cases, 5 cases ofUC and 5 cases of Crohn's disease showed significant differences in TJ permeability. The TJs from non-IBD patients were not penetrated by RR. In contrast, 55.3% of the TJ from inflammed areas and 9% from non-inflammed areas were permeable to RR in the colonic epithelium of UC patients. The colonic epithelium of Crohn's disease patients showed permeability to RR in 88.5% of the TJs in inflammed areas and 46.5% of the TJs in non-inflammed areas. Preliminary Western immunoblotting results showed changes in the phosphorylation of the Tl protein occludin in samples from inflammed areas. Conclusions: Our results suggest that increased TJ permeability in the colonic epithelia of Crohn's disease and UC patients may playa role in the development and progression of the disease. Alterations in the state of phosphorylation of TJ proteins and consequently, increased TJ permeability, may facilitate the entry of pathogens, contributing to epithelial barrier dysfunction and to the development of inflammation. This work was supported by NIH grant CA71l3 (to A.P.S.)
spreading and tight junction remodeling response by neighboring viable cells.
4265 PEROXYNITRITE-INDUCED NITRATION & OXIDATION IN CYTOSKELETAL INSTABILITY & LOSS OF INTESTINAL EPITHELIAL BARRIER FUNCTION (BF). Ali Banan, Sandeep Choudhary, Yang Zhang, Ali Keshavarzian, Rush Univ, Chicago, IL. Peroxynitrite (ONOO, a potent oxidizing agent) has been detected in the inflamed mucosa of several GI disorders (e.g. inflammatory bowel disease, IBD). Recent reports have proposed that ONOO is responsible for injury & loss of intestinal BF in IBD, but the underlying mechanism remains unexplored. We recently showed the importance of microtubule (MT) cytoskeleton in the maintenance of intact intestinal BF (Banan JPET, 291: 1075-1085, 1999). For the current investigation we hypothesized that ONOO-induced loss of intestinal BF is caused by nitration, oxidation, & disassembly / disruption of MT cytoskeleton. Methods: We evaluated the effects of several ONOO donors on epithelial BF, oxidation & nitration of tubulin (structural backbone of MT) & cytoskeletal disassembly / instability (n = 6 per group & *p<0.05). Human colonic cell (Caco-2) monolayers were preincubated in authentic ONOO or ONOO donors [morpholinosydnoimine (SIN-I) or S-3-nitroso-N-acetyl penicillamine (SNAP) + Xanthine (X) / X Oxidase] in the range of 0.01 - 5 mM :!: selective antioxidants [L-Cysteine, SOD (superoxide dismutase) or the inactive analouges DCysteine & iSOD, respectively]. Immunoblolling of fractionated polymerized tubulin (S2; index of stability) & the monomeric tubulin (SI; index of disruption) to detect oxidation, nitration, & disassembly of tubulin as well as immunofluorescent staining of MT followed by detailed evaluation by high-resolution laser confocal microscopy were performed. BF was determined by fluorescein sulfonic acid clearance. Results: ONOO or donors dose-dependently & significantly [1] t the stable S2 polymerized tubulin, [2] t the unstable Sl tubulin s, in concert, [3] t two measures of oxidative stress (fluorescence of dichlorofluorescein) & oxidation & nitration of tubulin (i.e., foot prints of ONOO generation). These compounds also dose-dependently & significantly disrupted BF & extensively disassembled / damaged MT. These effects were completely prevented by pretreatment with selective anti-oxidant scavengers: L-Cysteine or SOD but not their inactive analouges D-Cysteine or iSOD. Conclusions: Data demonstrate, for the first time, that oxidation, nitration, & disassembly of the MT cytoskeleton induced by ONOO causes the disruption of intestinal epithelial barrier function. Thus, the instability of this critical component may play an essential role in abnormal intestinal barrier function in inflammatory GI disorders associated with the presence of ONOO.
AGAAS03
4267 PROINFLAMMATORY CYTOKINES DISTURB ELECTROGENIC SODIUM ABSORPTION: A MECHANISM OF COLONIC DYSFUNCTION IN AN IBD MODEL. Christian Barmeyer, Ingo Grotjohann, Heinz Schmitz, Peter Scholz, Joachim Mankertz, Ernst O. Riecken, Michael Fromm, Joerg D. Schulzke, Univ Hosp Benjamin Franklin, Berlin, Germany; Schering AG, Berlin, Germany. We have recently shown that diarrhea in ulcerative colitis (UC) is caused by an impaired barrier function and that this defect is mediated by TNFO' and IFNy. An other mechanism contributing to diarrhea is impaired Na+ absorption in the distal colon. We studied the effect of these two cytokines as well as of IL-I 13 on active electrogenic Na + absorption in rat late distal colon in vitro. Using the same tissues we analyzed the mRNA expression of the 3 subunits of the epithelial Na+ channel (ENaC). Tissues were incubated in the Ussing chamber for 6 hours with TNFO'alone (loo ng/ml), a high dose combination of TNFO'+ IFNy (100 ng/ml + 100 Vlml), a low dose combination ofTNFO'+IFNy (10 ng/ml + 10 U/ml), IFNy alone (100 U/ml), and IL-If3 alone (10 ng/ml). Then, 3 nM aldosterone was added and short circuit current (lsc) was monitored for additional 8 hours. Finally, electrogenic Na+ absorption (JNa in p.moleh-1ecm-2) was quantified by addition of 10-4 M amiloride. SUbse~uently, the 0'-, 13- and y-subunit mRNA expression of the epithelial Na channel (ENaC) was detected by Northern blot analysis. As shown in the Table, treatment with either TNFO', TNFO'+ IFNy (high conc.) or IL-lf3 reduced JN a if compared to control. In parallel, Northern blot analysis revealed a lower RNA expression of ENaC 13- and y-subunits, whereas the a-subunit was unchanged. Neither TNFO'+ IFNy (low conc.) nor IFNy alone altered I N • or RNA expression of any of the 3 ENaC subunits. In conclusion: Proinflammatory cytokines TNFO' and IL-If3lead to impaired electrogenic Na" absorption in rat late distal colon as well as to a down-regulation of mRNA expression of the ENaC 13- and y-subunits, while that of the a-subunit was unchanged. IFN-y can intensify the effect of TNFO'. This may represent a distinct pathomechanism in colonic inflammation contributing to diarrhea as e.g. in IBD.
TNFu+IFNy
J", % ofcontrol
n
Pv,lu,
control
high cone.
lowcone.
TNFu
IFNy
IL.lp
13.4±1.1 (100) 11
1.8±0.5 14 8 <0.001
11.7±2.3 88 13 n.s.
5.9±1.1 44 10 <0.001
16.5±2.4 123 10
3.5±08 26 10 <0.001
n.s.
4266 INCREASED OXIDATION & NITRATION INJURY IN INTESTINAL MUCOSA OF PATIENTS WITH IBD AS DETECTED BY A HIGHLY SENSITIVE IMMUNOBLOT. Yang Zhang, Ali Banan, Todd M. Murphy, Sandeep Choudhary, Ali Keshavarzian, Rush Univ, Chicago, IL. Nitric oxide (NO) overproduction & generation of peroxynitrite (ONOO, a potent oxidizer) has been proposed to be responsible for mucosal injury in IBD disorders. As a highly toxic reactive species, ONOO has been suggested to indiscriminately attack biomolecules essential to mucosal function. However, the critical contribution of ONOO to the pathogenesis of IBD remains unclear. Aims: To determine [I] the native levels of oxidation I nitration in the intestinal mucosal biopsies of normal subjects; [2] whether increased oxidation (carbonylation) & nitrotyrosination (nitration, a foot print of ONOO generation) can be found in mucosal biopsies of IBD patients. Methods: We have developed a highly reliable, rapid & sensitive quantitative immunblot that can detect oxidative alterations in flash frozen mucosal biopsies [n= 9 - 16 I group, p<0.05, (*) denotes significance] from control (CON) & patients with active (a) or inactive (i) Colitis [Ulcerative Colitis (UC), Crohn s (CC) & Specific Colitis (SC)] using monoclonal anti-carbonyl & anti-nitrotyrosine antibodies that were conjugated to peroxidase by 3 protein cross-linking with 0.2% gluteraldehyde. Two-dimensional (2-D) densitometry was then performed to quantitate oxidative changes. Results: Endoscopic colonic mucosa from CON exhibited a low level of native carbonyl (5.6 :!: 1.2%) & nitration (l9.2 ::': 1.9%) immunoreactivity. In contrast, colonic mucosa of patients with active colitis showed (l2 & 4 fold, respectively) increased (*) levels of both carbonyl [aUC, 71 :!: 5.8%; aCC, 87.5 ::': 11.8%; aSC, 91.4 :!: 6.4%] & nitration [75 ::': 12%; 87 :!: 10%; 48 ::': 1.2%, respectively]. When colitis was in remission levels of nitration [iUC, 50 :!: 1.3%; iCC, 65 :!: 10%] & carbonyl [iUC, 52.1 ::': 5.7%; iCC, 22.4 ::': 7.4%] decreased (*). Conclusions: Data demonstrate, for the first time, [1] that abnormally high immunoreactive levels of carbonyl & nitrotyrosine moieties ( foot prints of ONOO) are present in the inflamed colonic mucosa of patients with active colitis. Thus, oxidative damage may be an essential underlying cause of mucosal injury in Ulcerative Colitis & Crohn s Disease. However, these alterations are not specific to such disorders, as they are also present in Specific Colitis. [2] Mucosal oxidation / nitration is a useful marker of assessing the degree of oxidative stress & may be used as an index of disease activity in IBD.
4268 INCREASED TIGHT JUNCTIONAL PERMEABILITY ASSOCIATED WITH CROHN'S DISEASE AND ULCERATIVE COLITIS. Vikram Barpujari, James J. Thornton, James M. Mullin, Hilary M. Clarke, Alejandro Peralta Soler, The Lankenau Hosp, Wynnewood, PA; The Lankenau Med Research Ctr, Wynnewood, PA. Aim: The intestinal epithelium acts as a barrier between two fluid compartments. During an inflammatory process, the intestinal epithelium must preserve its barrier function to maintain tissue homeostasis. Previous studies have shown abnormal intestinal permeability in patients with Crohn's disease and ulcerative disease. The purpose of this study was to assess the permeability of tight junctions (TJ) of the intestinal epithelium of patients with Crohn' s disease and ulcerative colitis (UC), applying an in situ method that uses an electron-dense dye and electron microscopy. Methods: Colon pinch biopsies were placed in chambers designed specifically for this study, with the mucosa facing up. The epithelium was exposed for 30 min at room temperature to 2.5% glutaraldehyde in O.IM sodium cacodylate buffer pH 7.4 containing 0.3% ruthenium red (RR). After rinsing, the epithelium was exposed to 2% osmium tetroxide and 0.3% RR for 30 min. Samples were rinsed and processed for electron microscopy. Ultrathin sections were cut perpendicular to the surface in a Reichert Ultracut ultramicrotome and photographed in a JEOL 1200 electron microscope, for quantitative analysis of TJ permeability. Results: Analysis of 4 non-IBD cases, 5 cases ofUC and 5 cases of Crohn's disease showed significant differences in TJ permeability. The TJs from non-IBD patients were not penetrated by RR. In contrast, 55.3% of the TJ from inflammed areas and 9% from non-inflammed areas were permeable to RR in the colonic epithelium of UC patients. The colonic epithelium of Crohn's disease patients showed permeability to RR in 88.5% of the TJs in inflammed areas and 46.5% of the TJs in non-inflammed areas. Preliminary Western immunoblotting results showed changes in the phosphorylation of the Tl protein occludin in samples from inflammed areas. Conclusions: Our results suggest that increased TJ permeability in the colonic epithelia of Crohn's disease and UC patients may playa role in the development and progression of the disease. Alterations in the state of phosphorylation of TJ proteins and consequently, increased TJ permeability, may facilitate the entry of pathogens, contributing to epithelial barrier dysfunction and to the development of inflammation. This work was supported by NIH grant CA71l3 (to A.P.S.)