Role of endotoxin in intestinal reperfusion-induced expression of E-selectin

Philippe Bauer, Janice M. Russell, and D. Neil Granger

Department of Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport, Louisiana 71130-3932


    ABSTRACT
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Products of enteric bacteria, including endotoxin [lipopolysaccharide (LPS)], have been implicated in the acute inflammatory responses elicited by ischemia and reperfusion (I/R) of the small intestine. The objective of this study was to assess the contribution of LPS to the increased E-selectin expression observed in the intestinal vasculature after I/R. The dual radiolabeled monoclonal antibody technique was used in LPS-sensitive (C3HeB/FeJ) and LPS-insensitive (C3H/HeJ) mice that were exposed to either exogenous LPS or to gut I/R (45 min ischemia, 5 h reperfusion). LPS elicited a dose-dependent (0.5-50 µg LPS/animal) increase in E-selectin expression (at 3 h) in LPS-sensitive mice, whereas LPS-insensitive mice were largely unresponsive. E-selectin expression was increased fivefold by I/R in the small bowel of both LPS-sensitive and -insensitive mice. These results indicate that, although exogenous LPS is capable of eliciting profound dose-dependent increases in E-selectin expression, endogenous LPS does not contribute significantly to I/R-induced expression of this endothelial cell adhesion molecule.

lipopolysaccharide; endotoxin-resistant mice; enteric bacteria; tumor necrosis factor-alpha


    INTRODUCTION
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Abstract
Introduction
Materials and methods
Results
Discussion
References

REPERFUSION OF ISCHEMIC tissues is associated with an enhanced production of inflammatory mediators, increased leukocyte trafficking, and endothelial barrier dysfunction in postcapillary venules. Data derived from both in vitro (16) and in vivo (6a, 9, 13) models of ischemia (hypoxia) and reperfusion (reoxygenation) (I/R-H/R) injury also reveal a role for endothelial cell adhesion molecules (CAM) in this disease process. This assertion is largely supported by experiments that demonstrate either an increased expression of adhesion molecules on endothelial cells exposed to I/R or H/R (21) or an attenuated leukocyte recruitment and/or injury response to I/R in animals that are pretreated with blocking monoclonal antibodies (MAb) to endothelial CAM (18, 26).

Endothelial cell monolayers exposed to H/R exhibit an increased expression of different adhesion glycoproteins, including E-selectin and intercellular adhesion molecule 1 (ICAM-1) (16). The H/R-induced upregulation of these endothelial CAM requires several hours for maximal expression and appears to be under transcriptional regulation. Although these in vitro models have provided valuable insight concerning the kinetics of endothelial CAM expression after H/R and the role of specific nuclear transcription factors in this response, the relevance of these observations to the mechanisms that regulate endothelial CAM expression in postischemic tissues remains unclear. A significant limitation of the in vitro models of I/R-induced inflammation is the inability to fully mimic the complex in vivo environment wherein the activation products (e.g., cytokines) of multiple auxilliary cell types (e.g., mast cells, macrophages) could exert a profound influence on endothelial CAM expression. The cytokines that are generated at accelerated rates by macrophages and mast cells in postischemic tissue are likely to contribute to the elevated endothelial CAM expression (31). The stimulus for I/R-induced cytokine release probably varies between tissues, but in the intestine endotoxins produced by enteric bacteria have been assigned an important role in eliciting both the local and distant inflammatory responses elicited by gut I/R (27, 30).

It is widely held that disruption of the intestinal epithelial lining by I/R results in the translocation of bacteria and bacterial products such as lipopolysaccharide (LPS) from the gut lumen to mucosal interstitium (28). Within the interstitial compartment (lamina propria), LPS can engage with receptors on macrophages to stimulate the production and release of cytokines, which in turn may act on the microvasculature to enhance endothelial CAM expression and the consequent recruitment and activation of leukocytes. Although exogenous LPS is known to increase the expression of endothelial CAM such as E-selectin in the small intestine (7), it remains unclear whether endogenous (luminally derived) LPS contributes to the increased endothelial CAM expression that is elicited by I/R. To address this issue, we measured the expression of E-selectin in the intestinal vasculature of genetically related strains of mice that are either sensitive (C3HeB/FeJ) or insensitive (C3H/HeJ) to exogenous LPS. The studies were predicated on the assumption that if endogenous LPS does indeed contribute to I/R-induced E-selectin expression, then LPS-insensitive mice should exhibit an attenuated E-selectin response (compared with LPS-sensitive mice) after I/R. An initial series of experiments was undertaken to verify that E-selectin upregulation in response to exogenous LPS differs between C3HeB/FeJ and C3H/HeJ mice.


    MATERIALS AND METHODS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Monoclonal antibodies. The MAb used for the in vivo assessment of E-selectin expression were 10E6, a binding rat IgG2a against mouse E-selectin (20), and P-23, a nonbinding murine IgG1 directed against human P-selectin (Pharmacia-Upjohn, Kalamazoo, MI) (19). The binding (10E6) and nonbinding (P-23) MAb were labeled with 125I and 131I, respectively (DuPont-New England Nuclear, Boston, MA) using the 1,3,4,6-tetrachloro-3alpha ,6alpha -diphenylglycouril (IODO-GEN) method (11). Briefly, iodogen (Pierce, Rockford, IL) was dissolved in chloroform at a concentration of 0.5 mg/ml, and 250 µl of this solution were placed in glass tubes and evaporated under nitrogen. A 250-µg sample of MAb was added to each iodogen-coated tube, and either 125I or 131I (1 µCi/µg protein) was added. The mixture was incubated on ice, with periodic stirring for 5-10 min. The total volume was brought to 2.5 ml by adding PBS (pH 7.4). Thereafter, the coupled MAb was separated from free 125I or 131I by gel filtration on a Sephadex PD-10 column (Pharmacia, Uppsala, Sweden). The column was equilibrated (50 ml) and then eluted (2.5, 2.5, 1, and 7 ml) with PBS containing 1% BSA. Four fractions were collected, the second 2.5 ml of which contained the radiolabeled MAb. Absence of free 125I or 131I was ensured by extensive dialysis of the protein-containing fraction. Less than 1% of the activity of the protein fraction was recovered from the dialysis fluid. Radiolabeled MAb were stored at 4°C and used within 1 (131I-P-23) or 2 mo (125I-10E6) after labeling.

Animal procedures. Six- to seven-week-old male LPS-sensitive (C3HeB/FeJ, n = 40) and LPS-insensitive (C3H/HeJ, n = 40) mice (Jackson Laboratory, Bar Harbor, ME), weighing 27 ± 0.4 g, were used in the radiolabeled antibody experiments. The mice were anesthetized subcutaneously with 150 mg/kg body weight of ketamine and 7.5 mg/kg of xylazine. The right jugular vein and right carotid artery were cannulated with polyethylene tubing (PE-10). To measure E-selectin expression, a mixture of 10 µg binding 125I-labeled anti-E-selectin MAb and a dose (0.5-5 µg) of nonbinding 131I-labeled MAb, adjusted to ensure a total 131I-injected activity of 500,000 ± 100,000 counts/min, were injected through the jugular vein catheter (total volume 200 µl). Mice were then heparinized with 50 IU heparin sodium in 0.2 ml saline. Blood samples (200 µl) were obtained through the carotid artery catheter 5 min after injection of the MAb mixture for measurement of plasma 125I and 131I activity (50 µl). Thereafter, an isovolemic blood exchange was rapidly performed with bicarbonate-buffered saline (6 ml) through the carotid artery catheter. Thoracic inferior vena cava was severed and flushed with bicarbonate-buffered saline (15 ml) through the carotid artery catheter. All the organs were harvested and weighed for radioactivity measurements. These experimental procedures were performed according to the criteria outlined by the National Institutes of Health and approved by the Louisiana State University Medical Center-Shreveport, Committee on Animal Care and Use.

Calculation of E-selectin expression. The method for calculation of E-selectin expression has been previously described (7). In brief, binding 125I-anti-E-selectin MAb and nonbinding 131I-MAb in different organs and in 50-µl aliquots of cell-free plasma were counted in a 14800 Wizard 3 gamma counter (Wallac, Turku, Finland), with automatic correction for background activity and spillover. The injected activity in each experiment was calculated by counting a 2-µl sample of the mixture containing the radiolabeled MAb. The radioactivities remaining in the tube used to mix the MAb and the syringe used to inject the mixture were substracted from the total calculated injected activity. The accumulated activity of E-selectin MAb in an organ was expressed as microgram of 125I-anti-E-selectin MAb per gram of tissue. E-selectin expression was calculated by substracting the accumulated activity of the nonbinding 131I-MAb (131I-P-23) from the activity of the binding 125I-anti-E-selectin MAb (125I-10E6). Previous studies have shown that MAb retain their functional activity after radioiodination as evidenced by a similar effectiveness of labeled and unlabeled MAb to block leukocyte adherence in rat mesenteric venules (22). In addition, we have shown that constitutive and endotoxin-induced expression of E-selectin is not detectable in the small intestine and other tissues of E-selectin-deficient mice, unlike their wild-type counterparts (7).

Experimental protocols. Two experimental protocols were employed to address the specific questions and study objectives previously outlined. The dual radiolabeled monoclonal antibody technique was used to quantify the expression of E-selectin in the intestinal vasculature and other vascular beds of LPS-sensitive (C3HeB/FeJ) and LPS-insensitive (C3H/HeJ) mice that were subjected to either exogenous LPS administration (3 h) or to gut I/R (45 min ischemia and 5 h reperfusion). To verify that LPS-sensitive and -insensitive mice respond in a differential manner to LPS, Escherichia coli LPS (serotype 0111:B4, Sigma Chemical, St. Louis, MO) was administered (in 0.5 ml isotonic saline) intraperitoneally at doses of 0 (saline vehicle), 0.5, 2.5, 5, and 50 µg per animal. Because previous work from our laboratory demonstrated that E-selectin is upregulated within 3 h after LPS administration (7), the mice were killed and tissue samples were harvested at 3 h for measurement of E-selectin expression.

To compare the E-selectin responses of LPS-sensitive and -insensitive mice to intestinal I/R, animals were anesthetized subcutaneously with 150 mg/kg body weight of ketamine and 7.5 mg/kg of xylazine. The hair was shaved from the abdomen. A 2-cm long midline laparotomy was performed, and the superior mesenteric artery (SMA) was found by deflecting the loops of intestine to the left with moist gauze swabs. The SMA was temporarily occluded by placing a micro-bulldog clamp at its origin from the aorta. Immediate blanching of the small intestine verified that the blood supply had been completely shut off. The abdomen was then covered with a moist gauze pad. After 45 min of ischemia the clamp was removed, and after verifying return of blood supply to the gut, 3 ml of 0.9% saline were infused into the peritoneal cavity, the laparotomy incision was closed, and the animals were allowed to awake. The mice subjected to sham SMA occlusion were anesthetized, and after laparotomy the SMA was isolated but not occluded. On the basis of preliminary experiments that revealed that 45 min of ischemia and 5 h of reperfusion elicit a significant increase in E-selectin, the mice were reanesthetized 5 h after the sham or actual SMA occlusion period and E-selectin was measured. A subgroup of mice had no laparotomy performed and served as controls.

Another series of 10 LPS-sensitive (C3HeB/FeJ) and 10 -insensitive (C3H/HeJ) mice, weighing 27 ± 0.5 g, with each group, consisting of 5 mice exposed to sham surgery and 5 to intestinal I/R, was studied to assess plasma endotoxin concentration, leukocytes counts, and tissue myeloperoxidase (MPO) activity in small bowel and lungs after 5 h of reperfusion.

Determination of plasma endotoxin. The chromogenic limulus amebocyte lysate (LAL) test (QCL-1000, BioWhittaker, Walkersville, MD) was used to measure plasma endotoxin levels. Briefly, plasma samples were collected in pyrogen-free tubes and immediately frozen at -85°C. The test samples were diluted 1:20 with LAL reagent water and heated at 70°C for 5 min to remove nonspecific inhibitors. Duplicate test samples (50 µl) were mixed and incubated in a pyrogen-free microplate at 37°C with LAL (50 µl) for 10 min and then mixed and incubated at 37°C with chromogenic substrate (100 µl) for 6 min. The reaction was stopped with 50 µl of 25% vol/vol glacial acetic acid in LAL water. The adsorbance was read immediately at 410 nm in a spectrophotometer (Dynatech MR 5000, Guernsey, Channel Islands, UK). Endotoxin concentration was calculated by substracting the mean absorbance of blanks from the mean absorbance value of standards (0.1 to 1.0 EU/ml E. coli endotoxin 0111:B4) and samples, using a method prescribed by the manufacturer.

Circulating leukocytes. The number of circulating leukocytes was determined from a 25-µl blood sample obtained from the carotid artery at 5 h of reperfusion. Leukocytes were stained by mixing the blood sample with 465 µl of 3% acetic acid and 10 µl of 1% crystal violet. Polymorphonuclear cells and mononuclear cells were counted with the aid of a Neubauer hematocytometer.

MPO activity assay. MPO activity, which is widely used to quantify neutrophil accumulation in tissues, was assessed using the O-dianisidine method (2, 32). Tissue samples that were harvested and immediately frozen (-85°C) at 5 h after reperfusion, were thawed, weighed, suspended (10% wt/vol) in 50 mM potassium phosphate buffer (KPi), pH 6.0, containing 0.5% hexadecyltrimethylammonium bromide buffer (0.1 g/20 ml KPi), and homogenized. One milliliter of the homogenate was sonicated three times for 10 s and microcentrifugated at 12,000 rpm for 10 min at 4°C. The reaction was started by mixing and incubating the supernatant (100 µl) at 20-25°C for 5 min with a solution composed of 2,900 µl of 50 mM KPi, 30 µl of 20 mg/ml O-dianisidine dihydrochloride, and 30 µl of 20 mM hydrogen peroxide. The reaction was stopped by adding 30 µl of 2% sodium azide. The change in absorbance was read at 460 nm at 5 min in a spectrophotometer (Hitachi U-2000, Hitachi Instruments, Dallas, TX), and MPO activity was expressed as the amount of enzyme necessary to produce a change in absorbance of 1.0 per minute per gram of wet weight of tissue.

Statistics. The data were analyzed using a one-way ANOVA with Scheffé's (post hoc) test (StatView 4.02 for Macintosh computers). All values are reported as means ± SE. Statistical significance was set at P < 0.05.


    RESULTS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

A dose-dependent increase in E-selectin expression was observed in the small bowel in LPS-sensitive but not LPS-insensitive mice over an LPS dose range of 0.5-50 µg/animal (Fig. 1). This dose-dependent response was also observed in large bowel and lungs (Fig. 2). However, in others organs, such as the heart and liver, peak expression of E-selectin was achieved at the lowest dose of LPS, suggesting a variation in the number and/or affinity of LPS receptors in different vascular beds. In LPS-insensitive mice the lowest dose (0.5 µg) of LPS did not elicit a significant response in any tissue. However, small but significant increases in E-selectin expression were observed at the highest LPS dose (50 µg) in liver (P = 0.002) and spleen (P = 0.0009). In LPS-treated animals we observed a peak of E-selectin expression at 3 h in the LPS-sensitive animals and no change at 1, 3, or 5 h in LPS-insensitive mice.


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Fig. 1.   E-selectin expression in small bowel after lipopolysaccharide (LPS) challenge at different doses (0-50 µg) in LPS-sensitive (C3HeB/FeJ) and -insensitive (C3H/HeJ) mice. * P < 0.05 vs. control. £ P < 0.0001 between 2 types of mice. MAb, monoclonal antibodies.


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Fig. 2.   E-selectin expression in large bowel (A), heart (B), lungs (C), and liver (D) 3 h after LPS challenge in LPS-sensitive (C3HeB/FeJ) and -insensitive (C3H/HeJ) mice. * P < 0.05 vs. control. £ P < 0.0001 between 2 types of mice.

On demonstrating that E-selectin expression is differentially upregulated by LPS in LPS-sensitive and -insensitive mice, we addressed the possibility that I/R elicits similarly different E-selectin responses in murine intestine. In LPS-sensitive mice gut I/R produced an increase in E-selectin expression that was five times the expression measured in control animals (ANOVA, P < 0.0001), 2-2.5 times the reponse detected in sham-operated animals (ANOVA, P < 0.0001), and about one-half the response that was elicited by 0.5 µg exogenous LPS. A quantitatively similar increase (5-fold) in E-selectin expression was noted in the postischemic intestine of LPS-insensitive mice (Fig. 3). After I/R peak of E-selectin expression was observed after 5 h of reperfusion in LPS-sensitive mice, and the same fivefold increase was observed at 3, 5, or 7 h of reperfusion in LPS-insensitive animals, suggesting that the similar responses cannot be ascribed simply to a difference in the time course of adhesion molecule expression between the two groups. No other organs were actually affected by I/R in both groups of animals.


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Fig. 3.   E-selectin expression in small bowel after 45 min of ischemia and 5 h of reperfusion (I/R) of superior mesenteric artery in LPS-sensitive (C3HeB/FeJ) and -insensitive (C3H/HeJ) mice. P < 0.005 vs. * control and/or dagger  sham in each group, respectively. No statistical difference between 2 types of mice.

Plasma endotoxin concentrations were measured in both LPS-sensitive and -insensitive mice at 5 h of reperfusion and compared with sham-operated animals. In neither strain of mice were we able to detect a significant level of endotoxin (within the LAL assay sensitivity range of 0.1-1 EU/ml), either following I/R or in the sham group. Circulating leukocyte counts and tissue MPO were also compared between the two groups at 5 h after reperfusion. A significant change in leukocyte count after I/R was noted only for neutrophils in the LPS-insensitive mice (4,488 ± 675 vs. 2,525 ± 367, P = 0.04). In both groups of animals I/R elicited an increased MPO activity in the small bowel (Fig. 4), with no differences between the two mouse strains.


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Fig. 4.   Myeloperoxidase (MPO) activity in small bowel (A) and lungs (B) after 45 min of ischemia and 5 h of reperfusion (I/R) in LPS-sensitive (C3HeB/FeJ) and -insensitive (C3H/HeJ) mice. * P < 0.05 vs. sham.


    DISCUSSION
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Abstract
Introduction
Materials and methods
Results
Discussion
References

The potential contribution of gut-derived endotoxin to the pathobiology of ischemic tissue injury was first proposed more than fifty years ago (12). Since that time there have been numerous reports describing bacterial translocation and endotoxin transfer across an injured mucosal membrane in postischemic intestine (14). The time course and magnitude of endotoxemia that result from intestinal I/R appear to vary according to species studied as well as the duration and/or severity of the ischemic insult (3, 25), with prolonged periods of complete ischemia more likely to result in endotoxemia. Some investigators have suggested that the inability to detect endotoxin in portal venous blood after I/R relates to the preferential clearance of such large molecules (mol wt >1 million) from the mucosal interstitium via the lymphatic system (6). Hence, it is often assumed that while systemic and portal venous blood levels of endotoxin may not rise substantially after I/R, inflammatory cells and endothelial cells within the mucosal interstitium are likely to be exposed to significant levels of the bacterial product.

In recent years there has been growing interest in defining the inflammatory actions of bacterial endotoxins, many of which mimic the responses observed in tissues exposed to I/R. Both exogenously administered endotoxin and I/R are known to recruit, prime, and activate neutrophils, activate the complement and clotting cascades, and elicit the production and release of cytokines from macrophages and endothelial cells (14, 29). An important physiological response of vascular endothelial cells challenged with either endotoxin or I/R is an increased surface expression of CAM. Monolayers of cultured endothelial cells exposed to endotoxin or H/R respond with an increased synthesis and expression of ICAM-1, vascular CAM-1, P-selectin, and E-selectin (1, 10, 34), all of which serve as ligands for adhesion receptors expressed on circulating leukocytes. These in vitro studies have been corroborated by reports describing increased mRNA levels for, and endothelial surface expression of, CAM such as ICAM-1 and E-selectin (5) in tissues exposed to either I/R or exogenous endotoxin. Support for a role for endogenous bacterial products such as endotoxin in the regulation of endothelial cell adhesion molecular expression is provided by a report describing a significant reduction in the expression of ICAM-1 in rat intestine decontaminated by treatment with oral antibiotics (17).

The fact that I/R and exogenous endotoxin are able to elicit similar inflammatory responses (especially in terms of endothelial CAM expression), coupled to evidence demonstrating elevated mucosal interstitial or plasma levels of endogenous endotoxin after gut I/R, has led to the proposal that endotoxin produced by enteric bacteria may mediate at least some inflammatory responses observed in the postischemic intestine. Support for this hypothesis is largely derived from studies demonstrating that gut decontamination with antibiotics blunts the rise in plasma endotoxin and tumor necrosis factor-alpha (TNF-alpha ) levels, as well as the local (gut) and distant (lung) tissue injury and inflammation elicited by intestinal I/R (27, 30). The objective of this study was to further assess the possibility that luminally derived endotoxin contributes to some components of the inflammatory response elicited by gut I/R, i.e., an accumulation of neutrophils and an increased expression of the endothelial cell adhesion molecule E-selectin. The role of endotoxin was addressed by comparing the responses of E-selectin expression in the intestine of endotoxin-sensitive (C3HeB/FeJ) and endotoxin-insensitive (C3H/HeJ) mice exposed to either exogenous endotoxin or gut I/R. E. coli LPS serotype 0111:B4 was selected for this study because this bacteria represents an important component of the intestinal microflora in these animals.

The endotoxin-insensitive (C3H/HeJ) mouse has been previously employed by several laboratories to address the role of LPS in different models of acute and chronic inflammation. These mice typically require nearly 100-fold higher amounts of endotoxin to induce lethality compared with their endotoxin-sensitive counterparts (24). The hyporesponsiveness of the C3H/HeJ mouse to LPS has been attributed to a profoundly diminished capacity of their macrophages to release TNF-alpha and interleukin-1 after LPS stimulation (23). This defect appears to result from an inhibition of LPS-induced cytokine production at both transcriptional and posttranscriptional levels (4). Our findings are consistent with this mechanism because we observed that LPS elicits a dose-dependent increase in E-selectin expression in different regional vascular beds of C3HeB/FeJ but not C3H/HeJ mice.

The results of this study also indicate that the increase in E-selectin expression elicited by 45 min of gut ischemia followed by 5 h of reperfusion is comparable to that induced 3 h after the intraperitoneal administration of 0.5 µg LPS. This suggests that nanogram levels of LPS would be required to gain access to the mucosal interstitium (from the gut lumen) to produce the E-selectin upregulation elicited by gut I/R. However, a comparison of the intestinal E-selectin responses to I/R between C3HeB/FeJ and C3H/HeJ mice failed to show a statistically significant difference. This observation bears directly on the issue of the role of endotoxin in mediating the inflammatory responses elicited by I/R in the gut. Our findings would therefore argue against luminally derived LPS as a major mediator of the increased expression of at least one of the endothelial CAM, namely E-selectin, that is observed in the postischemic intestine. This conclusion is further supported by our observation that the accumulation of neutrophils (MPO) in the gut after I/R also did not differ between LPS-sensitive and -insensitive mice.

The transcription-dependent upregulation of E-selectin that is elicited by I/R likely involves the participation of cytokines such as TNF-alpha and interleukin-1 because these agents are known to be released from postischemic tissues (15). Candidate molecules other than endotoxin that could contribute to the release of cytokines in the postischemic intestine include oxidants and nitric oxide. The production of oxidants is enhanced, whereas nitric oxide production is diminished, in vascular endothelial cells of the postischemic gut. The resulting imbalance between oxidant and nitric oxide generation has been proposed as a mechanism for the activation of nuclear transcription factors (e.g., NF-kappa B) that bind to and activate the promoter region of the gene encoding E-selectin (8). This activation of NF-kappa B can be elicited directly by the oxidant-nitric oxide inbalance or indirectly because of cytokine release from tissue macrophages. Thus it appears that I/R-induced E-selectin expression in intestinal vascular endothelial cells may occur via mechanisms that are independent of luminally derived bacterial endotoxin.


    ACKNOWLEDGEMENTS

We thank F. Jourd'heuil and Dr. R. M. Wolcott for their assistance with the endotoxin assays.


    FOOTNOTES

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant P01-DK-43785. P. Bauer is a recipient of a grant from Institut Lilly and Laboratoire L. Lafon in France.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: D. N. Granger, Dept. of Molecular and Cellular Physiology, LSU Medical Center, 1501 Kings Highway, PO Box 33932, Shreveport, LA 71130-3932.

Received 18 May 1998; accepted in final form 3 November 1998.


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Abstract
Introduction
Materials and methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 276(2):G479-G484
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