1Department of Medical Cell Biology, Biomedical Center, Uppsala University, Uppsala, Sweden; and 2Department of Physiology and Pharmacology, University of New South Wales, Sydney, Australia
Submitted 4 May 2005 ; accepted in final form 1 July 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
intracellular adhesion molecule 1; P-selectin; endotoxin; flurbiprofen; intravital microscopy
The NSAID indomethacin can cause severe mucosal injury. Asako et al. (1) demonstrated that clinically relevant concentrations of indomethacin induced leukocyte rolling and adhesion (but not emigration) through an leukotriene B4 (LTB4)-dependent mechanism. Wallace and co-workers then showed that the gastric injury induced by indomethacin in both the rat (25) and rabbit (23) could be markedly reduced by immunoneutralization of the adhesion molecule CD11/CD18. They also found that antibodies against the adhesion molecules ICAM-1 and to a lesser extent P-selectin reduced both mucosal injury and leukocyte adherence in the rat (25). Indomethacin has been shown to elevate the level of expression of both ICAM-1 and P-selectin (17, 18). Furthermore, depletion of circulating neutrophils with specific antiserum significantly reduced NSAID-induced injury in both the rat stomach (24) and small bowel (3).
Endotoxemia is a another pathophysiological situation in which the gastrointestinal tract is injured and adhesion molecule expression is dramatically enhanced. Panes et al. (19) found that administration of Salmonella abortus equii endotoxin to rats caused maximum upregulation of ICAM-1 mRNA 3 h after administration and maximum expression of ICAM-1 between 5 and 9 h after endotoxin exposure in both the stomach and intestine. Similar observations have been made for VCAM-1 and E-selectin in the mouse (16). This elevation in adhesion molecule expression is maintained well above basal levels for 24 h (19).
At present, there is no information on the level of expression of adhesion molecules in the different layers of the gut wall from the mucosa through to the serosa. The aim of this study was to measure the level of ICAM-1 and P-selectin expression within different transmural regions of the stomach and duodenum in control animals and after NSAID (flurbiprofen)- and endotoxin-induced upregulation. ICAM-1 and P-selectin expression were measured with radiolabeled antibodies (19). Because NSAID coupled with nitric oxide (NO) is less injurious to the gastrointestinal tract (26, 27), we also studied animals treated with NO-flurbiprofen to see whether the protective effect correlated with a reduction in adhesion molecule expression.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fifty-six male Sprague-Dawley rats (Möllegård, Denmark) or F1 hybrids of Lewis and Dark Agouti rats (Animal Department, Biomedical Center, Uppsala, Sweden) weighing 170240 g were kept under standardized conditions of temperature (2122°C) and illumination (12:12-h light-dark cycle) and were allowed free access to pelleted food (Ewos; Södertälje, Sweden). All experiments were approved by the Swedish Laboratory Animal Ethical Committee in Uppsala. Rats were fasted for 20 h before the experiments but had free access to water. They were anesthetized with thiobutabarbital sodium (120 mg/kg ip Inactin, Research Biochemicals; Natick, MA). Body temperature was maintained at 37 ± 0.5°C by a heating pad controlled by a rectal thermistor probe. A tracheostomy was performed, and a tracheal cannula was introduced to facilitate breathing.
Antibody Experiments
The right carotid artery and right jugular vein were cannulated for injection of antibodies and for blood sampling. At the end of the experiment, the animals were killed by exsanguination, and the stomach and small and large intestine were removed, washed free of contents, and treated as described below.
Labeling of ICAM-1 and P-Selectin with 125I and 131I
Monoclonal antibodies directed against ICAM-1 [IA29 (22)] or P-selectin [RMP-1 (28)] were labeled with 125I (DuPont NEN; Boston, MA), whereas isotype-matched nonbinding antibodies [P23 (14)] were labeled with 131I. (Antibodies were kindly supplied by Dr. D. Neil Granger, Louisiana State University, Shreveport, LA.)
Radioiodination was performed using the iodogen method, as previously described (19), in which 250 µg protein was incubated with 250 µCi of 125I or 131I for 5 min on ice in an iodogen-coated (125 µg) test tube. After incubation, the labeled antibodies were separated from the free 125I or 131I by gel filtration on a Sephadex PD-10 column (Pharmacia; Uppsala, Sweden). The column was equilibrated with 50 ml phosphate buffer containing 1% BSA. The labeled antibody was applied to the column and eluted with two fractions of 2.5 ml. The labeled antibody was contained in the second fraction. Absence of free 125I or 131I was ensured by dialysis, with <1% of the total activity appearing in the dialysate. SDS-PAGE showed normal heavy and light chain moieties of the expected molecular weights (19). The labeled antibodies were stored in 0.5-ml aliquots at 4°C for a maximum of 3 wk and were dialysed immediately before being used in each experiment.
Measurement of ICAM-1 and P-Selectin Expression
To measure ICAM-1 expression, a mixture of 10 µg of 125I-labeled ICAM-1 MAb (IA29), 230 µg of unlabeled IA29, and 5 µg of an isotype-matched nonbinding antibody (P-23) labeled with 131I were injected into the jugular vein catheter. These doses were determined by procedures described in a previous study (19). To measure P-selectin expression, a mixture of 10 µg of 125I -labeled P-selectin MAb (RMP-1) and 5 µg of 131I-labeled nonbinding MAb (P-23) were injected into a different group of rats. After the tracer injection, the animals were heparinized (3,000 IU/kg), and blood samples were taken via the carotid artery catheter at 2.5 and 5.0 min. At 5.0 min, the animal was exsanguinated via the carotid artery with a simultaneous infusion of bicarbonate buffer via the jugular vein. The vena cava was then severed, and the circulation was flushed via the carotid artery with 60 ml of buffer. Organs for study were then harvested and weighed.
Expression of ICAM-1 and P-Selectin Activity
The activity of 125I and 131I was determined using an LKB 1282 Compugamma (Wallac Oy; Turku, Finland). Samples were counted for sufficient time to obtain an accuracy of ±1%. The total activity injected (and the total nanograms of antibody injected) in each experiment was calculated by counting a 5-µl sample of the injectate. The activity remaining in the injection syringe was substracted from the total injected counts. The accumulated activity in the tissue was expressed as %antibody bound per gram tissue and was calculated as follows:
![]() |
![]() |
Division of the Gastrointestinal Tract
The gastrointestinal tract was divided into the forestomach, gastric corpus, gastric antrum, duodenum (proximal 10 cm of the small bowel), jejunum (50% of the remaining small bowel), ileum, cecum, and colon. Each section was washed cut open and blotted to remove water and mucus before being weighed and counted for 125I and 131I activity.
Separation of Gastroduodenal Walls Into Layers
Stomach. The gastric corpus was divided equally along the midline, one-half was pinned to a cork board mucosal surface uppermost, and the mucosa was removed by scraping with a scalpel. This procedure consistently removed the mucosa down to a resistant, shiny tissue plane, which histology revealed was the base of the gastric pits (see Histology). The removed mucosa was transferred to preweighed blotting paper and weighed immediately. The stomach was then reversed, and the muscularis layer was carefully removed with fine forceps and iris scissors under a dissecting microscope. This consistently divided the stomach at the clevage plane between the submucosa and muscularis, leaving the major vessels attached to the submucosa. The separated layers and the undivided half of the stomach were weighed, placed in counting tubes, and assessed for 125I and 131I activity.
Duodenum. The duodenum, which had been opened longitudinally and blotted dry, was divided into proximal 5 cm and distal 5 cm. The proximal portion was pinned to the corkboard mucosa uppermost and scraped with a scalpel until the procedure did not yield further tissue. Histology revealed that this procedure removed the mucosa down to the base of the villi (see Histology). The removed mucosa was transferred to preweighed blotting paper and weighed immediately. It was not possible to remove the muscularis from the duodenum, so only the two fractions were counted: the superficial mucosa and remaining deep mucosa and the submucosa plus muscularis. The distal duodenum was counted intact.
Histology
To determine the point of separation of the gut wall and the repeatability of this procedure, samples of the layers of the stomach and duodenum (excluding the mucosa, which was macerated during removal) and samples of the intact half of stomach and duodenum were fixed in 10% formalin, processed, embedded in wax, sectioned, and stained with haematoxylin. The point of separation of the mucosa represented by the scraping was assessed under a microscope (Laborlux 12, Leitz). These experiments were performed in a separate group of rats (n = 6) in which tissues were not harvested for counting 125I and 131I.
Experimental Treatments
Expression of ICAM-1 and P-selectin was induced with endotoxin (0.52.0 mg/kg ip S. abortus equi, Sigma) administered to anaesthetized animals 5 h before the injection of labeled antibodies. Another group was exposured to the NSAID flurbiprofen (20 mg/kg by gavage in carboxymethyl cellulose) 90 min before the injection of antibodies. Because the coupling of a NO group with a NSAID reduces its toxicity (10, 26, 27), we also investigated the effect of NO-flurbiprofen (30 mg/kg, Nicox; Milano, Italy) on adhesion molecule expression. The 30 mg/kg dose of NO-flurbiprofen contained the same amount of flurbiprofen as 20 mg/kg of the native compound (26). Control rats in both cases received vehicle alone.
Intravital Microscopy
The level of interaction of leukocytes with the endothelial wall in the mucosal microcirculation of the stomach of endotoxin-treated (S. abortus equi) rats (n = 10) was assessed by intravital microscopy as previously described by Holm-Rutili and Öbrink (11). Briefly, the forestomach was opened, and a segment of the stomach corpus was exposed and draped over a truncated conical pedestal, mucosal or serosal side up. The surface was bathed in warm (37°C) 0.9% NaCl solution by gently attaching a chamber with a pliable sealant (silicone grease, Dow Corning) to the mucosa or serosa. The studied area was viewed by transillumination with a xenon lamp (75 W) attached to a Leitz Ortholux microscope (Wetzlar, Germany). The images were projected onto a television screen (SONY PVM-90CE) via a television camera (Hamamatsu charge-coupled device camera C3077 with camera control C2400) and stored on a videorecorder (SONY V05630 Umatic) for later analysis. The area under observation was linearly magnified x25 by a Leitz water-immersion lens (sw 25/0.60), and the final magnification on the television screen was x780. Mucosal, submucosal, and muscularis venules up to a diameter of 20 µm were studied. Submucosal vessels were visible from the serosal side either directly or after the removal of 0.5 cm2 of the serosal muscle layer. Values were obtained for leukocyte adhesion and rolling as previously described (20). Leukocytes were regarded as adherent if they remained stationary for 30 s and longer and rolling if they moved with a lower velocity than red blood cells. The numbers of rolling or adherent leukocytes were measured during 2-min recordings and expressed as the numbers of rolling or adherent leukocytes per 100-µm length of vessel per minute.
Statistics
All values are expressed as means ± SE. Data were analyzed using one-way ANOVA and an unpaired t-test with a Bonferroni correction where multiple comparisons were made. Differences were regarded as significant at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Stomach. The stomach was divided along the midline, and one-half was separated into three layers. The superficial mucosa (designated mucosa) was removed by scraping, and this constituted 22 ± 1% of the total wall weight; the remaining mucosa plus submucosa (designated submucosa) was 56 ± 3%; and the muscularis was 22 ± 1% (Fig. 1). The average weight of stomach divided in this manner was 0.44 ± 0.02 g (n = 32). Histological analysis of the stomach after separation revealed that scraping consistently removed the mucosa down to the base of the gastric pits (see Fig. 1). Removal of the muscularis was complete and occurred at the natural cleavage plane between the muscularis and submucosa.
|
ICAM-1 Expression
Control and endotoxin-treated rats. The expression of ICAM-1 under control conditions and after upregulation with endotoxin is shown in Fig. 2. The level of expression in the superficial mucosa of the stomach was only 19% of that observed in the deep mucosa-submucosa when compared per gram of tissue. This difference was even more pronounced in the duodenum, where the level of expression in the mucosa was only 10% of that in the rest of the duodenal wall.
|
The expression of ICAM-1 along the gastrointestinal tract from the forestomach to colon is shown in Fig. 3 for control and endotoxin-treated animals. The level of expression in the gastric corpus (these data represent the intact stomach wall) was similar to that in the gastric antrum and also the colon. However, expression of ICAM-1 in all segments of the small bowel and cecum was significantly higher than in the stomach or colon. The relatively higher level of expression of ICAM-1 in the small bowel and cecum was observed under control conditions and also after endotoxemia. ICAM-1 expression in the duodenum was significantly (P < 0.05) higher than in the jejunum and ileum after exposure to endotoxin.
|
|
|
P-Selectin Expression
Control and endotoxin-treated rats. The expression of P-selectin under control conditions and after upregulation with endotoxin is shown in Fig. 4. In control animals, P-selectin expression in all layers of the stomach and duodenum was low (01.1 ng/g), indicating no constitutive expression of P-selectin. Endotoxin caused a modest but significant ( P < 0.05) upregulation of P-selectin in the mucosa of both the stomach and duodenum but a dramatic increase in the submucosa and muscularis layers of both organs. After endotoxin treatment, the level of expression of P-selectin per gram is very low in the mucosa (16% in the corpus and 5% in the duodenum) compared with the submucosa.
|
|
Intravital Microscopy
Endotoxin treatment caused upregulation of both ICAM-1 and P-selectin; however, the level of expression was much lower in the mucosa compared with the submucosa and muscularis. To see whether the lower level of expression resulted in less leukocyte-endothelial interactions, the levels of leukocyte rolling and adhesion were measured in the mucosa, submucosa, and muscularis of the rat stomach. Leukocyte-endothelial cell interactions were only studied in venules up to 20 µm in diameter because that was the upper limit of the vessel size observed in the superficial mucosa. Despite endotoxin treatment, the superficial mucosal venules did not show any adherent leukocytes and virtually no leukocyte rolling, with only two rolling leukocytes being observed in 1 of the 18 venules studied (Table 3). In contrast, venules in the submucosa and muscularis all showed consistent rolling, and the majority of venules studied had a consistent low level of adhesion (Table 3).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we measured adhesion molecule expression in different regions of the wall of the stomach and duodenum in control animals and after upregulation with either NSAID or endotoxin.
A key finding of this study was that ICAM-1 and P-selectin expression is consistently less in the superficial mucosa compared with the submucosa and muscularis. When expressed per gram of tissue, the level of ICAM-1 in the superficial gastric mucosa or pit region was 2025% of that observed in the submucosa or muscularis, and, for P-selectin, this value was 15%. In the duodenum, the difference was even more striking, with expression of ICAM-1 only 10% and P-selectin 5% in the villus region of the wall compared with the deeper layers. This difference was maintained when the animals were treated with endotoxin.
This analysis of intramural ICAM-1 and P-selectin expression depended on being able to reliably divide the gut wall into different regions. Histology revealed that the dissection technique in the stomach resulted in consistent removal of the gastric pits (crypts) down to the level of the gastric glands, and this superficial layer represented 22% of total wall weight. Removal of the muscularis was along a natural cleavage plane, was highly repeatable, and yielded 22% of total wall weight. In the duodenum, dissection removed the villi right down to their base at the level of the crypts, and this fraction represented 33% of total wall weight. There was excellent repeatability in the fractions removed by this method.
Vascular Architecture of the Gut Wall
A lower expression of ICAM-1 and P-selectin in the mucosa may reflect a lower density of blood vessels per unit weight of tissue in the superficial layers of the gut. There is no quantitative comparison of the vascularity of the different layers of the gut wall; however, the vascular architecture of the stomach and duodenum are well known. In the stomach, blood supply to the mucosa originates from arterioles that branch from the submucosal arterial plexus and break into capillaries at the most abluminal aspect of the mucosa. These capillaries form a rich plexus surrounding the gastric glands and track up to the mucosal surface, where they can be seen forming a polygonal array surrounding each gastric pit (8, 11). These capillaries coalesce at the luminal aspect of the mucosa into subepithelial venules that then drain into infrequently placed mucosal venules that have a mean separation distance of 350 µm in the rat (11). The mucosal venules pass directly down to submucosal venous plexus, receiving no further capillary tributaries en route (5). There is little doubt from vascular casts of the stomach wall that the gastric mucosa has a rich microvascular network. There is no evidence to suggest that the superficial mucosa is any less vascular than any other region of the stomach wall; however, its vessels are small, mainly capillaries, subepithelial postcapillary venules (15 µm in diameter), and relatively few mucosal collecting venules (
50 µm in diameter) (11). In contrast, the submucosa clearly contains many larger vessels, including submucosal arteries and veins.
In the small intestine, each villus is supplied by an arteriole or two that travels to the tip of the villus and breaks into a capillary plexus, which drains into a single venule that forms high in the villus at 80% of lamina proprial core height (2, 5). The capillaries in the villus below the point at which the venule forms originate from the capillary plexus that surrounds the adjacent intestinal glands. This portal arrangement provides blood to the lower part of the villus that has already passed through the pericryptal plexus. Vascular casts again indicate a rich vascular supply to the villi (6). Removal of the villi to the level of the crypts, as in the present study, yields a superficial mucosal sample with villus arterioles, capillaries, and villus venules. Like the stomach, the larger intramural arteries and veins in the small bowel are located in the submucosa.
Consideration of the architecture of the vessels in the wall of the stomach and small bowel clearly indicates that the superficial mucosa of both organs contains smaller vessels, in particular, a high proportion of capillaries. If the density of adhesion molecules expression was less in capillaries and small venules than in larger vessels, this would provide one explanation of why ICAM-1 and P-selectin levels are lower in the superficial mucosa.
Intravital Microscopy
A second explanation for the lower adhesion molecule expression in the superficial mucosa is the possibility that vessels in this region have a lower density of expression compared with the same size and type of vessel in a deeper layer of the gut wall. If this is true, one would expect there to be less leukocyte-endothelial interactions in comparable-sized vessels in the superficial mucosa compared with the submucosa or muscularis. To test this, we investigated leukocyte-endothelial interactions in the mucosa, submucosa, and muscularis of the stomach during endotoxemia. Vessels in the mucosa are small; the mean diameter of the 18 vessels studied was 14 µm. Size-matched vessels were chosen for comparison in both the submucosa and muscularis. In many previous studies of the gastric microcirculation, we have noted (unpublished observations) that in control animals there is no leukocyte rolling or adhesion in gastric mucosal blood vessels. In this study, both flurbiprofen and endotoxin treatment increased ICAM-1, whereas only endotoxin increased P-selectin. The values obtained for ICAM-1 expression after endotoxin administration were twice those recorded after flurbiprofen treatment. We therefore chose endotoxin-treated animals for intravital microscopy studies in which leukocyte-endothelial interactions in the mucosa were compared with those in the submucosa and muscularis to see whether the relatively higher levels of ICAM-1 and P-selectin expression caused leukocyte-endothelial interactions to occur in the mucosa. During endotoxemia, there is very consistent rolling of leukocytes in the submucosa and muscularis and a low level of adhesion. However, despite modest upregulation, the mucosa had only 1 vessel of 18 vessels studied that showed rolling and none that showed adhesion. The very low incidence of leukocyte-endothelial interaction is consistent with the lower level of expression of ICAM-1 and P-selectin observed in vessels of the superficial mucosa compared with same size vessels in the deeper layers of the gut wall.
The values shown in Table 3 are for vessels of approximately the same size in each transmural region. The 15- to 18-µm vessel size was dictated by the fact that the mucosa has mainly smaller vessels. However, the submucosa contains the majority of larger vessels in the gut wall, and these contributed to the measured levels of ICAM-1 and P-selectin so that the absolute values per gram of tissue (Table 1) were higher in the submucosa than the muscularis but the levels of rolling and adhesion in 15- to 18-µm vessels in each region were similar.
Effect of Flurbiprofen Versus NO-Flurbiprofen on the Gut Microcirculation
There is considerable evidence to suggest that leukocyte-endothelial cell interactions play a critical role in the pathophysiology of NSAID-induced gastropathy (3, 23, 24, 25). We investigated whether flurbiprofen preferentially increased the expression of ICAM-1 and P-selectin in the superficial mucosa, where NSAID injury is first observed macroscopically. Our results showed that ICAM-1 increased by 80% in the gastric mucosa. Because this increase was from a very low value of expression, even after upregulation the mucosa had only 50% of the level in the corpus as a whole or 30% of that recorded in the corpus submucosa. A similar observation was made in the duodenum, where the mucosal ICAM-1 expression increased significantly (73%) in response to flurbiprofen but even after this increase was still only 15% of the value in the remainder of the duodenal wall (Table 1).
Only the gastric corpus and proximal duodenum were divided into layers. When whole wall samples were analyzed from the forestomach right through to the colon, there was a consistent trend in that all flurbiprofen-treated tissues had higher values for ICAM-1 expression than control but only the gastric antrum and duodenum were significantly higher at the time point studied.
Whereas flurbiprofen treatment had a modest effect on ICAM-1 expression, it had no effect whatsoever on P-selectin levels in any organ studied.
Our findings of a NSAID-induced increase in ICAM-1 in the gastric mucosa agree with the findings of Morise et al. (17), who recorded an 40% increase in the whole stomach wall 1 and 3 h after oral indomethacin (20 mg/kg) treatment. However, they disagree with their finding that P-selectin was upregulated by NSAID. The difference might be a question of time of sampling because they only found a brief upregulation of P-selectin that was present 1 h after indomethacin treatment but absent at 3 h. Our measurements were performed 90 min after oral flurbiprofen treatment and therefore may have missed an earlier upregulation.
The values obtained for ICAM-1 expression in control animals in this study were higher than those obtained in the rat by Panes et al. (19) and Morise et al. (17) but similar to those obtained by Komatsu et al. (12) in the mouse. In the stomach, control values in this study were fourfold higher than the values obtained by Panes et al. (19) in the rat, whereas in the small bowel they were twice as high. However, in both studies, endotoxin caused a two- to threefold increase in ICAM expression. The differences in basal expression of ICAM may reflect differences in rats from different geographic locations that are bred and housed under slightly different conditions. The relative increase induced by endotoxin was similar despite the differences in basal expression.
The constituitive level of P-selectin expression in this study in control animals was similar to that obtained by Morise et al. (17) in the rat and slightly less than that obtained by Eppiheimer et al. (4) in the mouse. Whereas indomethacin was found to cause a small (25%) increase in P-selectin expression (17), flurbiprofen at normal therapeutic levels had no affect on P-selectin values.
Interestingly, NO-flurbiprofen had no affect on ICAM-1 or P-selectin values in either the mucosa or whole organ samples in any tissue studied. This is consistent with the observation that NO-NSAIDs are less injurious to the gastrointestinal tract than their parent compounds (15, 26).
In conclusion, this study suggests that the superficial gastric and duodenal mucosal microcirculation has a much lower density of ICAM-1 and P-selectin than observed in vessels in deeper layers of the gut, and this remains true even during stimulated upregulation with endotoxin. These results may explain the lack of leukocyte adhesion or reduced rolling observed in the superficial gastric mucosa. This implies that the circulation of the mucosa lining the gut can, by virtue of a deficiency of ICAM-1 and P-selectin, resist leukocyte rolling, adhesion, and extravasation. This may be an important property given the very inflammatory nature of the gut contents, e.g., bacterial products.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
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. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |