Experimental enteropathy in athymic and euthymic rats: synergistic role of lipopolysaccharide and indomethacin

Hideki Koga1, Kunihiko Aoyagi1, Takayuki Matsumoto1, Mitsuo Iida2, and Masatoshi Fujishima1

1 Second Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka 812-8582; and 2 Division of Gastroenterology, Department of Medicine, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of this study was to investigate the immunologic and microbiological bases of indomethacin enteropathy. Athymic nude and euthymic specific pathogen-free (SPF) rats were reared under conventional or SPF conditions. In each group, indomethacin was given intrarectally for 2 days. Indomethacin enteropathy was evaluated using a previously described ulcer index and tissue myeloperoxidase activity. Both euthymic and athymic nude rats developed intestinal ulcers to the same degree under conventional conditions but no or minimal ulcer under SPF conditions. Pretreatment of conventional rats with intragastric kanamycin sulfate, an aminoglycoside antibiotic, attenuated indomethacin enteropathy in a dose-dependent fashion. Interestingly, when lipopolysaccharide was injected intraperitoneally in kanamycin-pretreated rats, it fully restored enteropathy in these rats in a dose-dependent manner. We confirmed that kanamycin decreased the number of gram-negative bacteria and endotoxin concentration of the small intestine in a dose-dependent fashion. These results indicate that indomethacin enteropathy is bacteria dependent and does not require a T cell function. Synergy between indomethacin and bacterial lipopolysaccharide may play a major role in this enteropathy.

indomethacin-induced enteropathy; T cell function; intestinal flora; gram-negative bacteria; endotoxin


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INDOMETHACIN (Indo), a nonsteroidal anti-inflammatory drug (NSAID), is notorious for causing gastrointestinal injury (6). In experimental animals, Indo mainly has been administered orally or subcutaneously to examine the pathogenesis of gastric lesions (8, 20, 48, 51, 54). We have previously investigated the effects of intrarectal administration of Indo and reported that mucosal injury was detected predominantly in the small intestine (37). This Indo enteropathy mimics the inflammatory enteritis seen in Crohn's disease (4, 5, 16, 18, 35). Increased mucosal permeability, favorable effects of steroids and salicylazosulfapyridine, and preventive effects of polymeric or elemental diets are observed both in Indo enteropathy and in Crohn's enteritis. Indo enteropathy seems to be a useful model for Crohn's disease and can be expected to give clues to the pathogenesis of inflammatory bowel disease (3).

Various immunologic abnormalities have been reported in Crohn's disease. Lymphocytes, particularly T cells, have been described as being activated in inflamed tissues or serum in Crohn's disease (10, 12, 43). An interesting case of Crohn's disease with remission of bowel disease after human immunodeficiency virus infection has been reported (26). The authors suggested that a decreased CD4 lymphocyte count may have a favorable effect on Crohn's disease. Although Indo enteropathy has become an animal model for Crohn's disease, it is not known whether T lymphocytes contribute to the pathogenesis of Indo enteropathy. We have previously reported that FK-506 inhibits Indo enteropathy more than cyclosporin A or prednisolone (36). FK-506 is a strong immunosuppressive agent that inhibits T lymphocyte function (42, 59). It is thus necessary to clarify whether Indo enteropathy is mediated by T cell-dependent immunity.

Because FK-506 was described originally as an antibiotic, we decided to investigate whether intestinal bacteria affect Indo enteropathy. In 1969, Kent et al. (29) first reported the role of intestinal bacteria in Indo enteropathy. They found that a combination of three antibiotics inhibits the overgrowth of microorganisms in the small intestine and reduces the severity of Indo-induced ulcers. Several studies have shown that germ-free rats develop fewer intestinal lesions than conventional or specific pathogen-free (SPF) rats, suggesting a bacterial role in this enteropathy (39, 65). However, although Satoh et al. (50) confirmed the favorable effects of these same antibiotics, they concluded that antibiotics reduced gastrointestinal ulcers by their cytoprotective effect, not by their antibacterial actions. Thus the role of bacteria in Indo enteropathy remains controversial.

We report here evidence for a bacteria-mediated role in Indo enteropathy independent of T cell immunity.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male Wistar rats, weighing 150-250 g, aged 6-8 wk, and male F344/n rnu/rnu rats (athymic nude rats), weighing 150-200 g, aged 6-7 wk, purchased from Kyushu Animal (Tosu, Saga, Japan), were used in this study. The animals were housed in wire cages with a maximum of six animals per cage.

Care environments and administration of Indo. Athymic nude rats and Wistar rats were reared under SPF conditions; the animals were placed in sterile cages in laminar flow racks and were given an autoclaved diet and autoclaved tap water ad libitum. After 1-2 wk of care and maintenance, 24 mg/kg of Indo (Sigma Chemical) dissolved in carboxymethylcellulose were injected in the rectum for 2 days according to a previously described method (37). Other nude and Wistar rats were cared for under conventional conditions; they were placed in ordinary, nonsterile cages in nonsterile rooms and were given a nonsterile diet and water. The same dose of Indo was administered intrarectally to these rats after 1-2 wk of care.

Effects of antibiotics on Indo enteropathy. Wistar rats maintained under conventional conditions were treated with an aminoglycoside antibiotic, kanamycin sulfate (KM; 1, 10, and 100 mg/day; Meiji Seika Kaisha), which was dissolved in distilled water and administered daily via a metallic orogastric tube. After 7 days administration of KM, Indo was administered as described above.

Effects of lipopolysaccharide on Indo enteropathy in antibiotic-treated rats. Wistar rats cared for under conventional conditions were given 100 mg of KM daily via a metallic orogastric tube. After KM pretreatment for 7 days, 100, 300, or 1,000 µg of lipopolysaccharide (LPS) extracted from Escherichia coli K-235 (LPS; Sigma Chemical) per 100 g body weight were injected intraperitoneally. Twenty-four hours after LPS administration, Indo was administered as described above.

Evaluation of gastrointestinal lesions. Twenty-four hours after the second dose of Indo, all animals were killed by intraperitoneal injection of an overdose of sodium amobarbital. The stomach, small intestine, and cecum were removed, opened by a longitudinal incision, and pinned out on a wax block. The specimen was washed with saline, fixed in 10% Formalin for 2 days, and checked for any macroscopic change.

The stomach and the cecum were evaluated according to whether ulcers were present or not. Gastric or cecal ulcers were defined as ulcers of >3 mm in the greatest dimension. In the small intestine, longitudinal ulcers, which were defined as ulcers >10 mm long located on the mesenteric side of the lumen, were numbered, and their length was measured. Scattered ulcers also were assessed. The longitudinal ulcer index (UI) was calculated as the total length of longitudinal ulcers divided by the whole length of the small intestine.

Measurement of tissue myeloperoxidase activity. Tissue myeloperoxidase (MPO) activity was assayed in homogenates of the small intestinal mucosa with a spectrophotometric assay using 3,3',5,5'-tetramethylbenzidine (TMB; Sigma Chemical) as substrate (55). In brief, 100-200 mg of small intestinal tissue were homogenized in a ground-glass homogenizer in 10 vol of ice-cold PBS (20 mmol/l KHPO4; pH 7.4). The homogenate was centrifuged at 10,000 rpm for 15 min at 4°C, and the supernatant, which contained <5% total MPO activity, was discharged. The pellet was then rehomogenized in an equivalent volume of 50 mmol/l PBS (pH 6.0) containing 0.5% hexadecyltrimethylammonium (HETAB; Sigma Chemical) and 10 mmol/l EDTA. The pellet-HETAB suspension was freeze-thawed two times. Aliquots of the suspension were added to a solution containing 1.6 mmol/l TMB, 0.3 mmol/l H2O2, 80 mmol/l sodium phosphate buffer (pH 5.4), 8% N,N-dimethylformamide (Sigma Chemical), and 40% PBS in a total volume of 500 µl. The mixture was incubated for 3 min at 37°C and then was immersed in an ice bath. The reaction was terminated by the addition of 1.75 ml of 200 mM sodium acetate buffer (pH 3.0). One unit of enzyme activity was defined as the amount of MPO present that caused a change in absorbance of 1.0/min at 655 nm and 37°C. The data were corrected with tissue weight.

Tissue endotoxin assay. Small intestinal tissue (100-200 mg) was removed under sterile conditions and was homogenized in 10 vol of ice-cold PBS. Endotoxin concentration was assayed in the homogenates with a chromogenic endotoxin-specific test (Endospec SP; Seikagaku Kogyo). Briefly, this test consists of factor G-free Limulus-amebocyte lysate and a chromogenic substrate, t-butyloxycarbonyl-Leu-Gly-Arg-p-nitroanilide. The sample was added to a portion of Endospec SP test dissolved in 0.2 M Tris · HCl buffer (pH 8.0), and the mixture was incubated at 37°C for 30 min. Absorbance was measured at 545 nm after diazotization. The standard curve was plotted by using E. coli O111:B4 endotoxin in distilled water. The data were corrected with tissue weight.

Bacteriological examination of the small intestine. Small intestinal tissue (100-200 mg) was removed under sterile conditions and washed out rapidly and fully with PBS. The tissue was homogenized in 10 vol of PBS. The homogenates were diluted with Ringer solution. The aerobic plates were inoculated and incubated at 37°C for 24 h, and the anaerobic plates were examined after 48-72 h of incubation. All isolates were identified by standard methods, and surface counts at the various dilutions were performed. The colony counts were expressed as colony-forming units per gram of tissue.

Statistical analyses. Overall significance was determined by a one-way ANOVA for the length and number of small intestinal ulcers, MPO activity, endotoxin concentration, and bacteriological status and by chi 2 test for the incidence of cecal ulcers. P values <0.05 were considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Gastrointestinal damage under different care environments. Table 1 summarizes the intestinal lesions resulting under various conditions. Wistar rats cared for under conventional conditions developed a mean longitudinal ulcer length of 213.1 ± 16.8 mm in the small intestine, whereas under SPF conditions the mean ulcer length was significantly decreased to 7.0 ± 21.0 (SD) mm. The length of the small intestine of SPF-maintained Wistar rats was significantly longer than that of conventionally maintained Wistar rats. The number of small intestinal scattered ulcers was significantly less in SPF-maintained Wistar rats than in conventionally maintained Wistar rats. The mean UI for the small intestine, as shown in Fig. 1, was 26.7 ± 3.0 in conventionally maintained Wistar rats vs. 0.6 ± 1.8 in SPF-maintained Wistar rats. In athymic nude rats, conventionally maintained rats had a mean ulcer length of 210.8 ± 16.2 mm, whereas SPF-maintained rats developed no longitudinal ulcers. The degree of small intestinal longitudinal ulceration seen in conventionally maintained nude rats was as severe as in conventionally maintained Wistar rats (Fig. 1). In both Wistar and nude rats, the small intestinal ulcers were located in the midportion of the small intestine. In the stomach, no ulcers were induced in Wistar or nude rats under any condition. Cecal ulcers were produced in one of nine SPF-maintained Wistar rats and in none of the SPF-maintained nude rats. In contrast, cecal ulcers were seen in 5 of 10 conventionally maintained Wistar rats and 4 of 6 conventionally maintained nude rats. These results suggest that environmental factors are more important in Indo enteropathy than T cell-dependent immunity.

                              
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Table 1.   Indomethacin induced small intestinal ulcers in rats maintained under various conditions



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Fig. 1.   Under conventional conditions, the ulcer index in the small intestine is high in both Wistar and athymic nude rats. In contrast, when cared for under specific pathogen-free (SPF) conditions, both kinds of rats develop few or no longitudinal ulcers. Bars and error bars represent means and SD. * P < 0.001 vs. Wistar rats maintained under conventional conditions.

Effect of antibiotics on Indo enteropathy. Wistar rats cared for under conventional conditions with 1, 10, or 100 mg/day of KM pretreatment developed mean ulcer lengths of 155.3 ± 60.6, 89.3 ± 42.8, or 50.9 ± 41.8 mm in the small intestine, respectively. The length of the entire small intestine in rats pretreated with 1, 10, or 100 mg of KM was 902.3 ± 52.4, 930.4 ± 37.4, or 934.8 ± 78.8 mm, respectively (Table 2 and Fig. 2). Scattered ulcers in the small intestine were not affected by KM pretreatment. The incidence of gastric or cecal lesions was unchanged by KM pretreatment. As another marker of the intestinal inflammation, we evaluated MPO activity of the small intestine. MPO activity in rats without KM pretreatment measured 91.3 ± 20.9 (SE) U/g, whereas in rats with 1, 10, and 100 mg of KM pretreatment, MPO decreased by 37.0, 73.6, and 83.7%, respectively (Fig. 3). The UI of the small intestine and MPO activity was well correlated. These data show that KM inhibits Indo enteropathy in a dose-dependent fashion, suggesting a bacterial etiology for this enteropathy.

                              
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Table 2.   Effect of kanamycin sulfate on indomethacin-induced enteropathy



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Fig. 2.   Pretreatment with kanamycin sulfate (KM) prevents indomethacin (Indo) enteropathy in a dose-dependent manner. Furthermore, intraperitoneal injection of lipopolysaccharide (LPS) restores Indo enteropathy in a dose-dependent manner in KM-pretreated rats. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. Indo alone. dagger  P < 0.05 and dagger dagger P < 0.001 vs. 100 mg of KM-pretreated rats.


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Fig. 3.   Pretreatment with KM decreases tissue myeloperoxidase (MPO) activity in a dose-dependent manner. Change in MPO activity correlates well to that in the ulcer index shown in Fig. 2. * P < 0.01 and ** P < 0.005 vs. Indo alone.

Bacteriological status and endotoxin concentration of the small intestine. In Wistar rats cared for under SPF conditions, tissue endotoxin concentration measured only 0.03 ± 0.01 µg/g (Fig. 4). However, in rats cared for under conventional conditions, endotoxin remarkably increased to 98.4 ± 42.4 µg/g. With 1, 10, and 100 mg of KM pretreatment, endotoxin decreased by 33.2, 90.8, and 99.9%, respectively. Table 3 shows the results of tissue cultures of the small intestine in rats under various conditions. KM pretreatment decreased the number of gram-negative bacteria, especially Bacteroides species, in a dose-dependent manner. Gram-positive bacteria was killed with the least dose of KM. These findings support that endotoxin derived from gram-negative bacteria may play a key role in Indo enteropathy.


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Fig. 4.   Under SPF conditions, the guts of rats contain scarcely any endotoxin. Under conventional conditions, the small bowels of rats contain significant endotoxin. KM decreases tissue endotoxin in a dose-dependent manner. * P < 0.01 and ** P < 0.005 vs. sham. dagger  P < 0.05 vs. 1 mg of KM-pretreated rats.

                              
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Table 3.   Changes in bacterial flora of the small intestine in rats under various conditions

Effects of LPS on Indo enteropathy in antibiotic-treated rats. After Indo administration subsequent to one injection of 100, 300, or 1,000 µg/100 g body wt of LPS, KM-pretreated Wistar rats had mean ulcer lengths of 97.6 ± 36.4, 150.6 ± 26.9, or 206.7 ± 26.4 mm, respectively, in the small intestine. The length of the small intestine in the LPS-treated groups was 964.2 ± 86.3, 839.4 ± 139.3, or 888.9 ± 70.1 mm, respectively. The mean UI for the small intestine in the 1,000 µg LPS group returned to almost the same level seen in conventionally maintained rats without KM pretreatment, as shown in Fig. 2. The distribution of longitudinal ulcers was similar to that of conventionally maintained rats with no KM pretreatment and no LPS injection, i.e., the midportion of the small intestine. Wistar rats receiving 1,000 µg of LPS without Indo did not develop ulcers in the small intestine. The number of scattered ulcers in the small intestine was increased by LPS treatment in a dose-dependent manner. Rats treated with 1,000 µg of LPS developed many more scattered ulcers than LPS-untreated rats (41.7 ± 18.4 vs. 24.9 ± 13.2, P < 0.05). No gastric ulcers were seen in any group. Cecal ulcers developed in 4 of 5 rats receiving 100 µg of LPS and in 6 of 7 rats receiving 300 or 1,000 µg of LPS, but in only 3 of 10 rats not receiving LPS. Rats receiving 300 or 1,000 µg of LPS tended to have a higher incidence of cecal ulcers than LPS-untreated rats, but this difference was without statistical significance (P = 0.07). These findings suggest that LPS is involved in Indo enteropathy (Table 4).

                              
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Table 4.   Effect of lipopolysaccharide on indomethacin-induced gastroenteropathy in rats pretreated with kanamycin sulfate


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Indo, an analgesic-antipyretic and anti-inflammatory agent, was introduced in 1963 for the treatment of rheumatoid arthritis and related disorders. The toxicity of this drug often provokes many adverse effects: gastrointestinal ulcerations, pancreatitis, central nervous system disorders, and hematopoietic reactions. Therefore, Indo has been used to experimentally induce ulcerations of the stomach and the intestine in animals. In most previous reports, Indo has been given orally or subcutaneously. With these methods of Indo administration, gastric ulcers are prominent and have been regarded as more important than intestinal ulcers. However, intrarectal administration of Indo, which we previously described in detail (37), chiefly induces longitudinal ulcers in the small intestine, mimicking Crohn's disease.

Various reports have described mechanisms of Indo gastropathy. Indo inhibits cyclooxygenase activity, resulting in a decrease in endogenous cytoprotective prostaglandins and an increase in cytotoxic leukotrienes (32, 33, 46, 48, 63, 66). Prostaglandin E2 or 5-lipoxygenase inhibitor has been reported to prevent NSAID-induced gastric ulcers (47, 61). Some studies have shown that Indo provokes gastric hypermotility (40, 56, 57). Agents blocking peristalsis, such as neomycin and urethan, also prevent Indo-induced gastric lesions. Recently, much attention has been paid to the role of oxygen-derived free radicals in many disorders. Several investigations have shown that antioxidants such as superoxide dismutase have a preventive effect on NSAID-induced gastropathy (15, 57, 61). Thus Indo gastropathy is a multifactorial disorder. Although these mechanisms of Indo gastropathy in part can be applied to Indo enteropathy, the precise mechanisms underlying the latter still remain unresolved.

Athymic nude rats carry an autosomal recessive mutation, resulting in an absent thymus (21). The resulting severe T cell deficiency causes profoundly impaired T cell and T cell-dependent reactivity. Athymic nude rats characteristically contain few CD4+CD8+ intestinal intraepithelial lymphocytes (58). Normal B cell function has been observed in nude rats (9), and natural killer cell activity is also normal or higher than that in euthymic rats (14). Proliferation of intestinal mucosal mast cells, cytokine production, and the eosinophil response to parasites are also normal (2, 11, 41). Therefore, in some experiments, athymic nude rats have been used to investigate the role of T cells in the gastrointestinal tract (13, 17, 30, 38, 53). In both athymic nude rats and Wistar rats, conventionally maintained rats developed intestinal ulcers, whereas SPF-maintained rats did not. This suggests that Indo enteropathy is not related to T cell function but to environmental factors. The most important difference in environmental factors between conventional and SPF conditions is the presence of bacteria. We treated rats with KM before Indo administration to sterilize the intestine. KM, an aminoglycoside antibiotic, was first produced and isolated in 1957 and is useful for treating serious gram-negative bacterial infections. Orally administered KM is not absorbed, and its effect is limited to killing the gut flora. As expected, KM alone prevented Indo enteropathy in a dose-dependent manner in our study. Kent et al. (29) have reported that treatment with neomycin, polymyxin B, and bacitracin inhibits Indo enteropathy and suggested that intestinal flora play a role in the development of Indo enteropathy. However, because this combination of antibiotics kills various microorganisms, it is difficult to determine what organisms may play a pathogenetic role. Although Satoh et al. (50) also have suggested a role for bacteria in Indo enteropathy, they did not investigate this in detail. They did report that neomycin prevents Indo gastropathy not by its antibacterial action but by a cytoprotective effect. The favorable effect of oral KM in our study suggests a close relation between intestinal gram-negative bacteria and enteropathy. Actually, we confirmed that oral KM decreased the number of gram-negative bacteria and tissue endotoxin concentration in a dose-dependent manner. However, because antibiotics have various actions, a bacterial etiology for Indo enteropathy cannot be confirmed only by the preventive actions of KM. For example, neomycin is reported to accelerate orocecal transit in patients with hepatic cirrhosis (62), suggesting that neomycin increases intestinal and gastric motility (40).

To clarify the nature of bacterial involvement in Indo enteropathy, we injected LPS intraperitoneally before Indo administration in KM-pretreated rats because LPS is the major physiologically active substance derived from bacteria, especially the gram-negative bacteria frequently seen as part of the gut flora. We observed that LPS restored Indo enteropathy in a dose-dependent fashion. With 1,000 µg of LPS, Indo enteropathy returned almost to the level seen without KM treatment. This finding strongly suggests a role for bacteria, especially those containing LPS, in this enteropathy. In several knockout mouse models of inflammatory bowel disease, the presence of gut flora is necessary for the development of mucosal injury (34). Our results would support these previous studies.

Inhibition of cyclooxygenase is widely accepted as a major pharmacological effect of Indo, resulting in changes in prostanoids (32, 33, 48, 66). However, these changes in prostanoids may not affect the intestine directly, because Indo alone produced few ulcers in this study. This result suggests the presence of other pathways in forming intestinal ulcers. Our study demonstrates that intraperitoneal LPS and intrarectal Indo affected the small intestine. This phenomenon may support the importance of bile in Indo enteropathy, because Indo undergoes enterohepatic recirculation (19, 52) and because bile is rich in LPS (45). In Indo enteropathy, histological examination shows early infiltration of eosinophils and neutrophils (1). We had questioned why leukocyte infiltration and accumulation is induced by Indo administration despite Indo being an anti-inflammatory agent that can inhibit leukocyte accumulation. Because LPS is a strong chemotactic substance that promotes leukocyte accumulation (7, 24, 25, 31, 64), our observation that LPS is involved in Indo enteropathy may resolve this question. Although LPS also acts as a leukocyte stimulator (22, 23, 27, 49), our current study and a previous report (60) clarify that LPS alone does not induce enteropathy. Thus synergy between Indo and LPS may be essential for this enteropathy.

In this study, when the gut bacteria were killed with KM, the gut may have been exposed to a large amount of LPS, which is a constituent of gram-negative bacteria and which is released from killed bacteria. In the present study, however, KM treatment alone did not induce enteropathy. In addition, injection of purified LPS alone did not affect the intestine. This means that LPS alone may not induce enteropathy despite being a strong chemoattractant and proinflammatory agent. Although Indo alone did not affect the intestine in the absence of LPS, Indo did induce enteropathy in the presence of LPS. Thus our data agree with the idea that the synergistic action of Indo and LPS may play a key role in this enteropathy. The role of bacteria in Indo enteropathy, which has been indicated by previous reports (29, 50, 65), thus may be due to LPS.

It still remains unclear what factors play intermediate roles in the presence of both Indo and LPS. Considering the pharmacological actions of these two substances, this enteropathy may involve many inflammatory modulators, such as prostaglandins, leukotrienes, free radicals, and inflammatory cytokines. Some studies have suggested a microcirculatory disturbance in Indo enteropathy (28). Nygard et al. (44) showed that Indo augments LPS-induced procoagulant activity in cultured human umbilical vein endothelial cells and that Indo enteropathy may be relevant to microvascular injury and thrombosis. This may reinforce our results and hypothesis. Further investigations are required to elucidate the precise mechanism(s) of this experimental enteropathy.


    ACKNOWLEDGEMENTS

We thank Dr. Masanori Furuse for advice to measure myeloperoxidase activity and Dr. Kazutaka Makino for help with drug preparation.


    FOOTNOTES

Present address and address for reprint requests and other correspondence: H. Koga, Div. of Gastroenterology, Dept. of Medicine, Kawasaki Medical School, Matsushima 577, Kurashiki, Okayama 701-0192, Japan.

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.

Received 5 July 1998; accepted in final form 9 November 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Anthony, A., A. P. Dhillon, G. Nygard, M. Hudson, C. Piasecki, P. Strong, M. A. Trevethick, N. M. Clayton, C. C. Jordan, R. E. Pounder, and A. J. Wakefield. Early histological features of small intestinal injury induced by indomethacin. Aliment. Pharmacol. Ther. 7: 29-40, 1993[Medline].

2.   Arizono, N., T. Shiota, M. Yamada, Y. Matsumoto, H. Yoshikawa, S. Matsuda, and T. Tegoshi. Bromodeoxyuridine labeling studies on the proliferation of intestinal mucosal mast cells in normal and athymic rats. APMIS 98: 369-376, 1990[Medline].

3.   Banerjee, A. K., and T. J. Peters. Crohn's disease and NSAID enteropathy---a unifying model. Gastroenterology 99: 1190-1192, 1990[Medline].

4.   Banerjee, A. K., and T. J. Peters. Experimental non-steroidal anti-inflammatory drug-induced enteropathy in the rat: similarities to inflammatory bowel disease and effect of thromboxane synthetase inhibitors. Gut 31: 1358-1364, 1990[Abstract].

5.   Bjarnason, I., P. Williams, P. Smethurst, T. J. Peters, and A. J. Levi. Effect of non-steroidal anti-inflammatory drugs and prostaglandins on the permeability of the human small intestine. Gut 27: 1292-1297, 1986[Abstract].

6.   Bjarnason, I., G. Zanelli, T. Smith, P. Prouse, P. Williams, P. Smethurst, G. Delacey, M. J. Gumpel, and A. J. Levi. Nonsteroidal antiinflammatory drug-induced intestinal inflammation in humans. Gastroenterology 93: 480-489, 1987[Medline].

7.   Bozza, P. T., H. C. Castro-Faria-Neto, M. A. Martins, A. P. Larangeira, J. E. Perales, P. M. R. Silva, and R. S. B. Cordeiro. Pharmacological modulation of lipopolysaccharide-induced pleural eosinophilia in the rat; a role for a newly generated protein. Eur. J. Pharmacol. 248: 41-47, 1993[Medline].

8.   Brodie, D. A., P. G. Cook, B. J. Bauer, and G. E. Dagle. Indomethacin-induced intestinal lesions in the rat. Toxicol. Appl. Pharmacol. 17: 615-624, 1970[Medline].

9.   Brooks, C. G., P. J. Webb, R. A. Robins, G. Robinson, R. W. Baldwin, and M. F. W. Festing. Studies on the immunobiology of rnu/rnu "nude" rats with congenital aplasia of the thymus. Eur. J. Immunol. 10: 58-65, 1980[Medline].

10.   Brynskov, J., N. Tvede, C. B. Andersen, and M. Vilien. Increased concentrations of interleukin 1beta , interleukin-2, and soluble interleukin-2 receptors in endoscopical mucosal biopsy specimens with active inflammatory bowel disease. Gut 33: 55-58, 1992[Abstract].

11.   Candinas, D., S. Belliveau, N. Koyamada, T. Miyatake, P. Hechenleitner, W. Mark, F. H. Bach, and W. W. Hancock. T cell independence of macrophage and natural killer cell infiltration, cytokine production, and endothelial activation during delayed xenograft rejection. Transplantation 62: 1920-1927, 1996[Medline].

12.   Choy, M. Y., J. A. Walker-Smith, C. B. Williams, and T. T. MacDonald. Differential expression of CD25 (interleukin-2 receptor) on lamina propria T cells and macrophages in the intestinal lesions in Crohn's disease and ulcerative colitis. Gut 31: 1365-1370, 1990[Abstract].

13.   Collins, S. M., P. Blennerhassett, D. L. Vermillion, K. Davis, J. Langer, and P. B. Ernst. Impaired acetylcholine release in the inflamed rat intestine is T cell independent. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G198-G201, 1992[Abstract/Free Full Text].

14.   De Jong, W. H., P. A. Steerenberg, P. S. Ursem, A. D. M. E. Osterhaus, J. G. Vos, and E. J. Ruitenberg. The athymic nude rat. III. Natural cell-mediated cytotoxicity. Clin. Immunol. Immunopathol. 17: 163-172, 1980[Medline].

15.   Del Soldato, P., D. Foschi, G. Benori, and C. Scarpignato. Oxygen free radicals interact with indomethacin to cause gastrointestinal injury. Agents Actions 17: 484-488, 1986[Medline].

16.   Del Soldato, P., D. Foschi, L. Varin, and S. Daniotti. Indomethacin-induced intestinal ulcers in rats: effects of salicylazosulfapyridine and dexamethasone. Agents Actions 16: 393-396, 1985[Medline].

17.   D'Inca, R., P. Ernst, R. H. Hunt, and M. H. Perdue. Role of T lymphocytes in intestinal mucosal injury. Inflammatory changes in athymic nude rats. Dig. Dis. Sci. 37: 33-39, 1992[Medline].

18.   Drees, D. T., T. L. Robbins, and F. L. Crago. Effect of low-residue foods on indomethacin-induced intestinal lesions in rats. Toxicol. Appl. Pharmacol. 27: 194-199, 1974[Medline].

19.   Duggan, D. E., K. F. Hooke, R. M. Noll, and K. C. Kwan. Enterohapatic circulation of indomethacin and its role in intestinal irritation. Biochem. Pharmacol. 24: 1749-1754, 1975[Medline].

20.   Fang, W. F., A. Broughton, and E. D. Jacobson. Indomethacin-induced intestinal inflammation. Am. J. Dig. Dis. 22: 749-760, 1977[Medline].

21.   Festing, M. F. W., D. May, T. A. Connors, D. Lovell, and S. Sparrow. An athymic nude mutation in the rat. Nature 274: 365-366, 1978[Medline].

22.   Fittschen, C., R. A. Sandhaus, G. S. Worthen, and P. M. Henson. Bacterial lipopolysaccharide enhances chemoattractant-induced alastase secretion by human neutrophils. J. Leukoc. Biol. 43: 547-556, 1988[Abstract].

23.   Henricks, P. A., M. E. Van der Tol, R. M. Thyssen, B. S. Van Asbeck, and J. Verhoef. Escherichia coli lipopolysaccharides diminish and enhance cell function of human polymorphonuclear leukocytes. Infect. Immun. 41: 294-301, 1983[Medline].

24.   Issekutz, A. C., and S. Bhimji. Role for endotoxin in the leukocyte infiltration accompanying Escherichia coli inflammation. Infect. Immun. 36: 558-566, 1982[Medline].

25.   Issekutz, T. B., A. C. Issekutz, and H. Z. Movat. The in vivo quantitation and kinetics of monocyte migration into acute inflammatory tissue. Am. J. Pathol. 103: 47-55, 1981[Abstract].

26.   James, S. P. Remission of Crohn's disease after human immunodeficiency virus infection. Gastroenterology 95: 1667-1669, 1988[Medline].

27.   Kaku, M., K. Yagawa, S. Nagao, and A. Tanaka. Enhanced superoxide anion release from phagocytes by muramyl dipeptide or lipopolysaccharide. Infect. Immun. 39: 559-564, 1983[Medline].

28.   Kelly, D. A., C. Piasecki, A. Anthony, A. P. Dhillon, R. E. Pounder, and A. J. Wakefield. Focal reduction of villous blood flow in early indomethacin enteropathy: a dynamic vascular study in the rat. Gut 42: 366-373, 1998[Abstract/Free Full Text].

29.   Kent, T. H., R. M. Cardelli, and F. W. Stamler. Small intestinal ulcers and intestinal flora in rats given indomethacin. Am. J. Pathol. 54: 237-249, 1969[Medline].

30.   Khan, I., and S. M. Collins. Expression of cytokines in the longitudinal muscle myenteric plexus of the inflamed intestine of rat. Gastroenterology 107: 691-700, 1994[Medline].

31.   Kopaniak, M. M., A. C. Issekutz, and H. Z. Movat. Kinetics of acute inflammation induced by E. coli in rabbits. Quantitation of blood flow, enhanced vascular permeability, hemorrhage, and leukocyte accumulation. Am. J. Pathol. 98: 485-498, 1980[Abstract].

32.   Levine, R. A., S. Petokas, J. Nandi, and D. Enthoven. Effects of nonsteroidal, antiinflammatory drugs on gastrointestinal injury and prostanoid generation in healthy volunteers. Dig. Dis. Sci. 33: 660-666, 1988[Medline].

33.   Ligumsky, M., M. Sestieri, F. Karmeli, J. Zimmerman, E. Okon, and D. Rachmilewitz. Rectal administration of nonsteroidal antiinflammatory drugs. Effect of rat gastric ulcerogenicity and prostaglandin E2 synthesis. Gastroenterology 98: 1245-1249, 1990[Medline].

34.   MacDonald, T. T. Cytokine gene deleted mice in the study of gastrointestinal inflammation. Eur. J. Gastroenterol. Hepatol. 9: 1051-1055, 1997[Medline].

35.   Matsumoto, T., M. Iida, F. Kuroki, K. Hizawa, H. Koga, and M. Fujishima. Effects of diet on experimentally induced intestinal ulcers in rats: morphology and tissue leukotrienes. Gut 35: 1058-1063, 1994[Abstract].

36.   Matsumoto, T., M. Iida, S. Nakamura, K. Hizawa, F. Kuroki, and M. Fujishima. Preventive effect of immunosuppressive agents against indomethacin-induced small intestinal ulcers in rats. Dig. Dis. Sci. 39: 787-795, 1994[Medline].

37.   Matsumoto, T., M. Iida, S. Nakamura, F. Kuroki, K. Hizawa, and M. Fujishima. An animal model of longitudinal ulcers in the small intestine induced by intracolonically administered indomethacin in rats. Gastroenterol. Jpn. 28: 10-17, 1993[Medline].

38.   McKay, D. M., M. Benjamin, M. Baca-Estrada, R. D'Inca, K. Croitoru, and M. H. Perdue. Role of T lymphocytes in secretory response to an enteric nematode parasite. Studies in athymic rats. Dig. Dis. Sci. 40: 331-337, 1995[Medline].

39.   Melarange, R., G. Moore, P. R. Blower, M. E. Coates, F. W. Ward, and V. Ronaasen. A comparison of indomethacin with ibuprofen on gastrointestinal mucosal integrity in conventional and germ-free rats. Aliment. Pharmacol. Ther. 6: 67-77, 1992[Medline].

40.   Mersereau, W. A., and E. J. Hinchey. Neomycin prevents indomethacin-induced gastric peristalsis and mucosal injury in the rat. Can. J. Physiol. Pharmacol. 67: 1029-1032, 1989[Medline].

41.   Milbourne, E. A., and M. J. Howell. Eosinophilia in nude rats and nude mice after injection with Fasciola hepatica or injection with its E/S antigens. Int. J. Parasitol. 27: 1099-1105, 1997[Medline].

42.   Morris, R. E., E. G. Hoyt, M. P. Murphy, and R. Shorthouse. Immunopharmacology of FK-506. Transplant. Proc. 21: 1042-1044, 1989[Medline].

43.   Mueller, C., P. Knoflach, and C. C. Zielinski. T-cell activation in Crohn's disease. Increased levels of soluble interleukin-2 receptor in serum and in supernatants of stimulated peripheral blood mononuclear cells. Gastroenterology 98: 639-646, 1990[Medline].

44.   Nygard, G., M. Hudson, G. Mazure, A. Anthony, A. P. Dhillon, R. E. Pounder, and A. J. Wakefield. Procoagulant and prothrombotic responses of human endothelium to indomethacin and endotoxin in vitro. Scand. J. Gastroenterol. 30: 25-32, 1995[Medline].

45.   Osnes, T., P. Kierulf, A. G. Skar, R. Øvstebø, and M. Osnes. Quantification of lipopolysaccharides in human bile with or without gram-negative bacteria. Scand. J. Gastroenterol. 27: 453-458, 1992[Medline].

46.   Rainsford, K. D. Mucosal lesions induced in the rat intestinal tract by the anti-inflammatory drug, Wy-41,770, a weak inhibitor of prostaglandin synthesis, contrasted with those from the potent prostaglandin inhibitor, indomethacin. Toxicol. Pathol. 16: 366-375, 1988[Medline].

47.   Rainsford, K. D. Leukotrienes in the pathogenesis of NSAID-induced gastric and intestinal mucosal damage. Agents Actions 39: C24-C26, 1993[Medline].

48.   Robert, A. An intestinal disease produced experimentally by a prostaglandin deficiency. Gastroenterology 69: 1045-1047, 1975[Medline].

49.   Sasada, M., M. J. Pabst, and R. B. Johnston, Jr. Activation of mouse peritoneal macrophages by lipopolysaccharide alters the kinetic parameters of the superoxide-producing NADPH oxidase. J. Biol. Chem. 258: 9631-9635, 1983[Abstract/Free Full Text].

50.   Satoh, H., P. H. Guth, and M. I. Grossman. Role of bacteria in gastric ulceration produced by indomethacin in the rat: cytoprotective action of antibiotics. Gastroenterology 84: 483-489, 1983[Medline].

51.   Satoh, H., I. Inada, T. Hirata, and Y. Maki. Indomethacin produces gastric antral ulcers in the refed rats. Gastroenterology 81: 719-725, 1981[Medline].

52.   Schneider, H. T., B. Nuernberg, K. Dietzel, and K. Brune. Biliary elimination of non-steroidal anti-inflammatory drugs in patients. Br. J. Clin. Pharmacol. 29: 127-131, 1990[Medline].

53.   Soda, K., S. Kawabori, N. Kanai, J. Bienenstock, and M. H. Perdue. Steroid-induced depletion of mucosal mast cells and eosinophils in intestine of athymic nude rats. Int. Arch. Allergy Immunol. 101: 39-46, 1993[Medline].

54.   Stewart, T. H. M., C. Hetenyi, H. Rowsell, and M. Orizaga. Ulcerative enterocolitis in dogs induced by drugs. J. Pathol. 131: 363-378, 1980[Medline].

55.   Suzuki, K., H. Ota, S. Sasagawa, T. Sakatani, and T. Fujikura. Assay method for myeloperoxidase in human polymorphonuclear leukocytes. Anal. Biochem. 132: 345-352, 1983[Medline].

56.   Takeuchi, K., H. Niida, T. Ohuchi, and S. Okabe. Influences of urethane anesthesia on indomethacin-induced gastric mucosal lesions in rats. Dig. Dis. Sci. 39: 2536-2542, 1994[Medline].

57.   Takeuchi, K., S. Ueki, and S. Okabe. Importance of gastric motility in the pathogenesis of indomethacin-induced gastric lesions in rats. Dig. Dis. Sci. 31: 1114-1121, 1986[Medline].

58.   Takimoto, H., T. Nakamura, M. Takeuchi, Y. Sumi, T. Tanaka, K. Nomoto, and Y. Yoshikai. Age-associated increase in number of CD4+CD8+ intestinal intraepithelial lymphocytes in rats. Eur. J. Immunol. 22: 159-164, 1992[Medline].

59.   Tocci, M. J., D. A. Matkovich, K. A. Collier, P. Kwok, F. Dumont, S. Lin, S. Degudicibus, J. J. Siekierka, J. Chin, and N. I. Hutchinson. The immunosuppressant FK506 selectively inhibits expression of early T cell activation genes. J. Immunol. 143: 718-726, 1989[Abstract/Free Full Text].

60.   Turek, J. J., and I. A. Schoenlein. Indomethacin-induced gastrointestinal ulcers in rats: effects of dietary fatty acids and endotoxin. Prostaglandins Leukot. Essent. Fatty Acids 48: 229-232, 1993[Medline].

61.   Vaananen, P. M., J. B. Meddings, and J. L. Wallace. Role of oxygen-derived free radicals in indomethacin-induced gastric injury. Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G470-G475, 1991[Abstract/Free Full Text].

62.   Van Thiel, D. H., S. Fagiuoli, H. I. Wright, M. C. Chien, and J. S. Gavaler. Gastrointestinal transit in cirrhotic patients: effect of hepatic encephalopathy and its treatment. Hepatology 19: 67-71, 1994[Medline].

63.   Wagner, K. A., J. Nandi, R. L. King, and R. A. Levine. Effects of nonsteroidal antiinflammatory drugs on ulcerogenesis and gastric secretion in pylorus-ligated rat. Dig. Dis. Sci. 40: 134-140, 1995[Medline].

64.   Weg, V. B., D. T. Walsh, L. H. Faccioli, T. J. Williams, M. Feldmann, and S. Nourshargh. LPS-induced 111In-eosinophil accumulation in guinea-pig skin: evidence for a role for TNF-alpha. Immunology 84: 36-40, 1995[Medline].

65.   Weissenborn, U., S. Maedge, D. Buettner, and K. F. Sewing. Indomethacin-induced gastrointestinal lesions in relation to tissue concentration, food intake and bacterial invasion in the rat. Pharmacology 30: 32-39, 1985[Medline].

66.   Whittle, B. J. R. Temporal relationship between cyclooxygenase inhibition, as measured by prostacyclin biosynthesis, and the gastrointestinal damage induced by indomethacin in the rat. Gastroenterology 80: 94-98, 1981[Medline].


Am J Physiol Gastroint Liver Physiol 276(3):G576-G582
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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