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 |
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 |
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 |
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
2 test for the incidence of
cecal ulcers. P values <0.05 were
considered significant.
 |
RESULTS |
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.

View larger version (23K):
[in this window]
[in a new window]
|
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.

View larger version (34K):
[in this window]
[in a new window]
|
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.
P < 0.05 and
 P < 0.001 vs. 100 mg of KM-pretreated rats.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
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.

View larger version (16K):
[in this window]
[in a new window]
|
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. P < 0.05 vs. 1 mg
of KM-pretreated rats.
|
|
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).
View this table:
[in this window]
[in a new window]
|
Table 4.
Effect of lipopolysaccharide on indomethacin-induced gastroenteropathy
in rats pretreated with kanamycin sulfate
|
|
 |
DISCUSSION |
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 |
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 1
, 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