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Infection of intestinal epithelial cells with enteropathogenic Escherichia coli activates iron regulatory processes
GASTROENTEROLOGY Vol. 118, No.4
A796 AGA ABSTRACTS
4237
4239
SERUM LEVELS OF SOLUBLE INTERCELLULAR ADHESION MOLECULE·! (SICAM.!) CORRELATE WITH SEVERITY OF IL· EITIS IN EXPERIMENTAL CROHN'S DISEASE: A NOVEL MARKER OF SMALL INTESTINAL INFLAMMATION. Jesus Rivera-Nieves, R. Cartland Bums, Christopher A. Moskaluk, Theresa
INFECTION OF INTESTINAL EPITHELIAL CELLS WITH ENTEROPATHOGENIC ESCHERICHIA COLI ACTIVATES IRON REGULATORY PROCESSES.
T. Pizarro, Klaus F. Ley, Fabio Cominelli, Univ of Virginia, Charlottesville, VA. The lack of a reliable serologic marker of chronic small intestinal inflammation has been a limitation in assessing disease severity in both experimental models and patients with Crohn's ileitis. Serum amyloid A (SAA), interleukin-6 (IL-6) and sICAM-l have been previously proposed as markers of chronic colonic inflammation. The aim of this study was to investigate whether the aforementioned factors may predict severity of small intestinal inflammation in the SAMP1IYit and the TNF.1ARE mouse models of ileitis. Both models closely resemble human Crohn's disease and show histological features of patchy mucosal inflammation with transmural involvement and granuloma formation. Blood samples were obtained from both SAMP1IYit and TNF.1ARE mice and serum separated and immediately frozen at -70·C for later assay. Circulating SAA, IL-6 and sICAM-l were analyzed by specific ELISA. SAA and IL-6 levels did not correlate with the severity of ileitis in SAMPllYit mice. Conversely, serum levels of slCAM were elevated in mice between 30 and 40 weeks of age (n=4) in association with the onset of spontaneous ileitis (median = 252, range 202-310 ng/ml) compared to healthy control mice (n=4) «150 mg/ml, p <0.01). Levels of slCAM progressively increased up to 70 weeks of age (n=4) in correlation with disease severity (median=463, range 367-558, p < 0.01 vs controls). TNF.1ARE mice which develop a more severe form of chronic ileitis by 8 weeks of age (n=5) showed higher slCAM-1levels in correlation with disease severity (median=680, range=335-1025) compared to wild type healthy mice «150 mg/mI, p < 0.001). Our results show that SAA and IL-6 serum levels did not correlate with severity of ileitis in SAMP1IYit mice. In contrast, slCAM-l levels correlated higlhy with the severity of chronic small intestinal inflammation in two models of experimental Crohn's ileitis. We conclude that slCAM levels may represent an excellent marker of chronic small intestinal inflammation. Consequently, circulating sICAM-1 can potentially serve as both a marker of disease activity and in the assessment of response to therapy.
4238 PROSTANOIDS IN HUMAN COLONIC MUCOSA: EFFECTS OF INFLAMMATION ON PGE z RECEPTOR EXPRESSION. James K. Roche, Rosana Cosme, Darienne Lublin, Vivian Takafuji, Kevin Lynch, Univ of Virginia Health Sci Ctr, Charlottesville, VA. Although the tissue concentration of PGEz is heightened 3-fold or more during mucosal inflammation, the cellular targets of prostanoids in human mucosa and the resulting changes in cell physiology have not been fully explored. We used a panel of immunoglobulin and mRNA probes in order to localize and quantitate the four member EP family of prostanoid receptors for binding PGEz to cells of histologically normal and inflamed human colonic mucosa, and then examined prostanoid-induced changes in mucosal lymphocyte function. EP4 alone was expressed on lamina propria mononuclear cells (fig 1). Dual immunostaining in situ identified the CD3+ T lymphocyte as a major EP4 receptor-bearing cell in normal mucosa. Flow cytometry of isolated cells showed that 19.2% of lamina propria mononuclear cells were EP4 +, and almost 30% of these were CD3 +. In situ hybridization with digoxygenin-labelled RNA probes largely confirmed this localization. During inflammation, mucosal T lymphocytes showed a marked enhancement in EP4 immunoreactive receptor protein. Computer-assisted densitometry of single cells demonstrated an increase in fluorescence intensity from 4.8± 1.8 to 8.6:t 1.8 (p < 0.04) (fig 2). The effects of PGE z included a 35% reduction in T lymphocyte IL-2 secretion. The number of COX 1+ lamina propria cells nearly doubled during inflammation; expressed a T lymphocyte marker; but retained an unchanged quantity of immunoreactive COX 1 protein per cell. A major perturbation in the number and distribution of PGEz receptors and enzymes for prostanoid synthesis occurs in chronic inflammation of the colon, with consequences for mucosal T lymphocyte function.
11
,------:-::-:-:-:~ L P ~1C
10
~ 2.
Inc rea llr In U ', (,1111'1'",.n
b, T ttlle In ulC'. roUIL,=---
Ivana Simonovic, Maria Konstantinou, Nina Brown, William Walden, Gail Hecht, Univ of Illinois, Chicago, IL. Iron is believed to play an important role in host defense during bacterial infection. Defense mechanisms involving iron include production of hydroxyl radicals, which can be toxic to microbes, production of cytokines by activating the transcription factor NF-KB and sequestration of iron in ferritin, thus depriving pathogens of an essential element for growth. The molecular mechanism by which iron levels are regulated in eukaryotic cells is through iron regulatory proteins (IRP). IRP are a family of RNA binding proteins that post-transcriptionally regulate expression of genes encoding proteins involved in iron storage and transport. Regulation of gene expression by IRP is mediated through an mRNA sequence element, the iron responsive element (IRE), to which IRP bind. The aim of this study was to determine the impact of infection by an enteric bacterial pathogen, enteropathogenic E. coli (EPEC), on iron regulatory processes in intestinal epithelial cells. For these studies, human intestinal epithelial, T84 or Caco-Z, cells were infected with EPEC or non-pathogenic E. coli. Infection with EPEC caused a 3-4 fold increase in IRE binding activity of IRPs by 15 min post-infection, whereas non-pathogenic E. coli failed to elicit this response. Increased IRE binding persisted for 45 min then gradually returned to basal levels by 3hr postinfection. The rapid increase in IRE binding suggests that signaling mechanisms targeted at IRPs, as opposed to iron availability, may be responsible for the initial increase in IRE binding activity. To examine a potential consequence of increased IRE binding in response to infection, alterations in the level of transferrin receptor (TfR) expression were assessed by immunoblot analysis. Interestingly, EPEC upregulated transferrin receptor (TfR) expression by 3h (2 fold) which plateaued by 6h at a 3 fold increase. This level of expression persisted for at least 24h post-infection. In summary, infection of intestinal epithelial cells by an enteric bacterial pathogen, EPEC, but not non-pathogenic E. coli, rapidly increases IRE binding activity. One potential consequence of this event is the upregulation of TfR expression which would increase the host cell capacity for iron uptake. We speculate that EPEC-induced changes in intestinal epithelial cell iron metabolism may provide a mechanism of host defense by activating NF-KB thus increasing cytokine production, producing toxic free radicals, and/or by sequestering essential iron from microbes. 4240
CYCLOOXYGENASE (COX)·2·DERIVED PROSTAGLANDIN n, IS AN IMPORTANT, EARLY, ANTI-INFLAMMATORY SIGNAL IN EXPERIMENTAL COLITIS. Maureen N. Ajuebor, John L. Wallace, Univ of Calgary, Calgary, AB, Canada. Background: Prostaglandin (PG) synthesis, particularly via COX-2, is markedly up-regulated in inflammatory bowel disease (IBD). The ability of NSAIDs and COX-2 inhibitors to exacerbate IBD suggests that PGs are important anti-inflammatory mediators in this context. PGDz has recently been suggested to be primarily derived from COX-2 and to be an important anti-inflammatory signal in experimental pleuritis (Nature Medicine 1999; 5: 698-701). In this study, we have investigated the possiblity that PGDz , derived from COX-2, plays an important role in down-regulating colonic inflammation in the rat. Methods: Colitis was induced in male, Wistar rats by intracolonic administration of trinitrobenzene sulfonic acid (TNBS). At various times thereafter (3-24 h), rats were sacrificed (n2=5 per group), colonic tissue was excised and PGDz synthesis and myeloperoxidase (MPO) activity were measured, the latter as an index of granulocyte infiltration. Results: Within 3 h, TNBS administration had elicited a >3fold increase in colonic PGDz synthesis. At this timepoint, no significant changes in colonic granulocyte number had occurred. Treatment with a selective COX-2 inhibitor, celecoxib (10 mg!kg), or indomethacin (10 mg/kg) immediately after TNBS abolished the increase in PGDz synthesis and resulted in more than a doubling of tissue MPO activity at the 3 h timepoint. On the other hand, intracolonic administration of PGDz (100 p,g!kg)at 1 and 4 h post-TNBS resulted in a significant reduction of colonic MPO activity at the 8 h timepoint (from 24 ± 3 U/mg in vehicle-treated to 13 ± I U/mg in PGDz-treated; p<0.05). Conclusions: These results show that induction of colitis with TNBS results in a rapid increase in PGDz synthesis, and that this occurs via COX-2. Moreover, PGDz exerts antiinflammatory effects, markedly suppressing granulocyte infiltration into the colon. The ability of NSAIDs and selective COX-2 inhibitors to exacerbate colitis may be due, at least in part, to suppression of PGD 2 synthesis.
A796 AGA ABSTRACTS
4237
4239
SERUM LEVELS OF SOLUBLE INTERCELLULAR ADHESION MOLECULE·! (SICAM.!) CORRELATE WITH SEVERITY OF IL· EITIS IN EXPERIMENTAL CROHN'S DISEASE: A NOVEL MARKER OF SMALL INTESTINAL INFLAMMATION. Jesus Rivera-Nieves, R. Cartland Bums, Christopher A. Moskaluk, Theresa
INFECTION OF INTESTINAL EPITHELIAL CELLS WITH ENTEROPATHOGENIC ESCHERICHIA COLI ACTIVATES IRON REGULATORY PROCESSES.
T. Pizarro, Klaus F. Ley, Fabio Cominelli, Univ of Virginia, Charlottesville, VA. The lack of a reliable serologic marker of chronic small intestinal inflammation has been a limitation in assessing disease severity in both experimental models and patients with Crohn's ileitis. Serum amyloid A (SAA), interleukin-6 (IL-6) and sICAM-l have been previously proposed as markers of chronic colonic inflammation. The aim of this study was to investigate whether the aforementioned factors may predict severity of small intestinal inflammation in the SAMP1IYit and the TNF.1ARE mouse models of ileitis. Both models closely resemble human Crohn's disease and show histological features of patchy mucosal inflammation with transmural involvement and granuloma formation. Blood samples were obtained from both SAMP1IYit and TNF.1ARE mice and serum separated and immediately frozen at -70·C for later assay. Circulating SAA, IL-6 and sICAM-l were analyzed by specific ELISA. SAA and IL-6 levels did not correlate with the severity of ileitis in SAMPllYit mice. Conversely, serum levels of slCAM were elevated in mice between 30 and 40 weeks of age (n=4) in association with the onset of spontaneous ileitis (median = 252, range 202-310 ng/ml) compared to healthy control mice (n=4) «150 mg/ml, p <0.01). Levels of slCAM progressively increased up to 70 weeks of age (n=4) in correlation with disease severity (median=463, range 367-558, p < 0.01 vs controls). TNF.1ARE mice which develop a more severe form of chronic ileitis by 8 weeks of age (n=5) showed higher slCAM-1levels in correlation with disease severity (median=680, range=335-1025) compared to wild type healthy mice «150 mg/mI, p < 0.001). Our results show that SAA and IL-6 serum levels did not correlate with severity of ileitis in SAMP1IYit mice. In contrast, slCAM-l levels correlated higlhy with the severity of chronic small intestinal inflammation in two models of experimental Crohn's ileitis. We conclude that slCAM levels may represent an excellent marker of chronic small intestinal inflammation. Consequently, circulating sICAM-1 can potentially serve as both a marker of disease activity and in the assessment of response to therapy.
4238 PROSTANOIDS IN HUMAN COLONIC MUCOSA: EFFECTS OF INFLAMMATION ON PGE z RECEPTOR EXPRESSION. James K. Roche, Rosana Cosme, Darienne Lublin, Vivian Takafuji, Kevin Lynch, Univ of Virginia Health Sci Ctr, Charlottesville, VA. Although the tissue concentration of PGEz is heightened 3-fold or more during mucosal inflammation, the cellular targets of prostanoids in human mucosa and the resulting changes in cell physiology have not been fully explored. We used a panel of immunoglobulin and mRNA probes in order to localize and quantitate the four member EP family of prostanoid receptors for binding PGEz to cells of histologically normal and inflamed human colonic mucosa, and then examined prostanoid-induced changes in mucosal lymphocyte function. EP4 alone was expressed on lamina propria mononuclear cells (fig 1). Dual immunostaining in situ identified the CD3+ T lymphocyte as a major EP4 receptor-bearing cell in normal mucosa. Flow cytometry of isolated cells showed that 19.2% of lamina propria mononuclear cells were EP4 +, and almost 30% of these were CD3 +. In situ hybridization with digoxygenin-labelled RNA probes largely confirmed this localization. During inflammation, mucosal T lymphocytes showed a marked enhancement in EP4 immunoreactive receptor protein. Computer-assisted densitometry of single cells demonstrated an increase in fluorescence intensity from 4.8± 1.8 to 8.6:t 1.8 (p < 0.04) (fig 2). The effects of PGE z included a 35% reduction in T lymphocyte IL-2 secretion. The number of COX 1+ lamina propria cells nearly doubled during inflammation; expressed a T lymphocyte marker; but retained an unchanged quantity of immunoreactive COX 1 protein per cell. A major perturbation in the number and distribution of PGEz receptors and enzymes for prostanoid synthesis occurs in chronic inflammation of the colon, with consequences for mucosal T lymphocyte function.
11
,------:-::-:-:-:~ L P ~1C
10
~ 2.
Inc rea llr In U ', (,1111'1'",.n
b, T ttlle In ulC'. roUIL,=---
Ivana Simonovic, Maria Konstantinou, Nina Brown, William Walden, Gail Hecht, Univ of Illinois, Chicago, IL. Iron is believed to play an important role in host defense during bacterial infection. Defense mechanisms involving iron include production of hydroxyl radicals, which can be toxic to microbes, production of cytokines by activating the transcription factor NF-KB and sequestration of iron in ferritin, thus depriving pathogens of an essential element for growth. The molecular mechanism by which iron levels are regulated in eukaryotic cells is through iron regulatory proteins (IRP). IRP are a family of RNA binding proteins that post-transcriptionally regulate expression of genes encoding proteins involved in iron storage and transport. Regulation of gene expression by IRP is mediated through an mRNA sequence element, the iron responsive element (IRE), to which IRP bind. The aim of this study was to determine the impact of infection by an enteric bacterial pathogen, enteropathogenic E. coli (EPEC), on iron regulatory processes in intestinal epithelial cells. For these studies, human intestinal epithelial, T84 or Caco-Z, cells were infected with EPEC or non-pathogenic E. coli. Infection with EPEC caused a 3-4 fold increase in IRE binding activity of IRPs by 15 min post-infection, whereas non-pathogenic E. coli failed to elicit this response. Increased IRE binding persisted for 45 min then gradually returned to basal levels by 3hr postinfection. The rapid increase in IRE binding suggests that signaling mechanisms targeted at IRPs, as opposed to iron availability, may be responsible for the initial increase in IRE binding activity. To examine a potential consequence of increased IRE binding in response to infection, alterations in the level of transferrin receptor (TfR) expression were assessed by immunoblot analysis. Interestingly, EPEC upregulated transferrin receptor (TfR) expression by 3h (2 fold) which plateaued by 6h at a 3 fold increase. This level of expression persisted for at least 24h post-infection. In summary, infection of intestinal epithelial cells by an enteric bacterial pathogen, EPEC, but not non-pathogenic E. coli, rapidly increases IRE binding activity. One potential consequence of this event is the upregulation of TfR expression which would increase the host cell capacity for iron uptake. We speculate that EPEC-induced changes in intestinal epithelial cell iron metabolism may provide a mechanism of host defense by activating NF-KB thus increasing cytokine production, producing toxic free radicals, and/or by sequestering essential iron from microbes. 4240
CYCLOOXYGENASE (COX)·2·DERIVED PROSTAGLANDIN n, IS AN IMPORTANT, EARLY, ANTI-INFLAMMATORY SIGNAL IN EXPERIMENTAL COLITIS. Maureen N. Ajuebor, John L. Wallace, Univ of Calgary, Calgary, AB, Canada. Background: Prostaglandin (PG) synthesis, particularly via COX-2, is markedly up-regulated in inflammatory bowel disease (IBD). The ability of NSAIDs and COX-2 inhibitors to exacerbate IBD suggests that PGs are important anti-inflammatory mediators in this context. PGDz has recently been suggested to be primarily derived from COX-2 and to be an important anti-inflammatory signal in experimental pleuritis (Nature Medicine 1999; 5: 698-701). In this study, we have investigated the possiblity that PGDz , derived from COX-2, plays an important role in down-regulating colonic inflammation in the rat. Methods: Colitis was induced in male, Wistar rats by intracolonic administration of trinitrobenzene sulfonic acid (TNBS). At various times thereafter (3-24 h), rats were sacrificed (n2=5 per group), colonic tissue was excised and PGDz synthesis and myeloperoxidase (MPO) activity were measured, the latter as an index of granulocyte infiltration. Results: Within 3 h, TNBS administration had elicited a >3fold increase in colonic PGDz synthesis. At this timepoint, no significant changes in colonic granulocyte number had occurred. Treatment with a selective COX-2 inhibitor, celecoxib (10 mg!kg), or indomethacin (10 mg/kg) immediately after TNBS abolished the increase in PGDz synthesis and resulted in more than a doubling of tissue MPO activity at the 3 h timepoint. On the other hand, intracolonic administration of PGDz (100 p,g!kg)at 1 and 4 h post-TNBS resulted in a significant reduction of colonic MPO activity at the 8 h timepoint (from 24 ± 3 U/mg in vehicle-treated to 13 ± I U/mg in PGDz-treated; p<0.05). Conclusions: These results show that induction of colitis with TNBS results in a rapid increase in PGDz synthesis, and that this occurs via COX-2. Moreover, PGDz exerts antiinflammatory effects, markedly suppressing granulocyte infiltration into the colon. The ability of NSAIDs and selective COX-2 inhibitors to exacerbate colitis may be due, at least in part, to suppression of PGD 2 synthesis.