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STOMACH
Gastric damage and granulocyte infiltration induced by
indomethacin in tumour necrosis factor receptor 1 (TNF-R1)
or inducible nitric oxide synthase (iNOS) deficient mice
M H L P Souza, H Paula Lemos, R B Oliveira, F Q Cunha
...............................................................................................................................
See end of article for
authors’ affiliations
.......................
Correspondence to:
Dr M H L P Souza,
Departamento de
Fisiologia e Farmacologia,
Universidade Federal do
Ceara´, Rua Cel. Nunes de
Melo, 1127, Rodolfo
Teo´filo, Fortaleza-CE,
Brasil; souzamar@ufc.br
Accepted for publication
12 November 2003
.......................
Gut 2004;53:791–796. doi: 10.1136/gut.2002.012930
Background: Tumour necrosis factor a(TNF-a) is involved in non-steroidal anti-inflammatory drug induced
gastropathy. Nitric oxide (NO) is a mediator of gastrointestinal mucosal defence but, paradoxically, it also
contributes to mucosal damage.
Aims: We optimised the C57BL/6 mouse model of indomethacin induced gastropathy to evaluate the role
of TNF-aand inducible nitric oxide synthase (iNOS) generated NO in gastric damage and granulocyte
infiltration using tumour necrosis factor receptor 1 (TNF-R12/2) or iNOS (iNOS2/2) deficient mice.
Methods: Different doses of indomethacin (2.5, 5, 10, 20 mg/kg) were administered and animals were
assessed 6, 12, or 24 hours later. Gastric damage was measured by the sum of all erosions in the gastric
mucosa, and gastric granulocyte infiltration was determined by myeloperoxidase (MPO) activity. Other
groups of wild-type mice received thalidomide, dexamethasone, fucoidin, L-NAME, or 1400W, and then
indomethacin was administered. Additionally, indomethacin was administered to TNF-R12/2or
iNOS2/2. Gastric damage and MPO activity were evaluated 12 hours later.
Results: Indomethacin induced dose and time dependent gastric damage and increase in MPO activity in
wild-type mice, with the greatest effect at a dose of 10 mg/kg and after 12 hours. Treatment with
thalidomide, dexamethasone, or fucoidin reduced gastric damage and MPO activity induced by
indomethacin. After indomethacin administration, TNF-R12/2had less gastric damage and MPO activity
than controls. Genetic (knockout mice) or pharmacological (1400W and L-NAME) inhibition of iNOS
activity reduced indomethacin induced gastric damage, despite no reduction in MPO activity.
Conclusion: TNF-a, acting via TNF-R1, is involved in indomethacin induced gastric damage and
granulocyte infiltration. Furthermore, iNOS generated NO is involved in gastric damage induced by
indomethacin.
Non-steroidal anti-inflammatory drugs (NSAIDs) are
some of the most widely used drugs in the world.
1
NSAID induced gastric damage is the major side effect
associated with usage of this type of drug. Although the
mechanism of NSAID induced gastropathy is generally
believed to be related to the ability of these agents to inhibit
gastric prostaglandin generation,
1
there is a great deal of
evidence to suggest that neutrophils are important in the
pathogenesis of the gastric damage induced by NSAIDs.
226
It is becoming increasingly appreciated that tumour
necrosis factor a(TNF-a) plays a critical role in NSAID
induced gastric injury by modulating neutrophil infiltra-
tion.
45
The activities of TNF-aare mediated by two distinct
cell surface receptors: TNF receptor 1 (TNF-R1) and TNF
receptor 2 (TNF-R2).
7
Several studies have shown that TNF-R1
is the dominant signalling receptor for the inflammatory
effect of TNF-a.
7
However, the role of TNF-R1 in NSAID
induced gastric damage has not been studied.
Nitric oxide (NO) is a crucial mediator of gastrointestinal
mucosal defence but, paradoxically, it also contributes to
mucosal damage.
8
This can be illustrated by the ability of
different NO concentrations to produce completely opposite
effects in the same tissue.
9
In general, the neuronal and
endothelial nitric oxide synthase (NOS) isoforms produce
low amounts of NO.
9
In contrast, the inducible form of NOS
(iNOS) produces NO in higher quantities.
9
Piotrowski et al
showed that when animals received an ulcerogenic dose of
indomethacin, there was a 12-fold increase in gastric
epithelial expression of iNOS activity compared with controls,
and this increase was positively correlated with damage to
the epithelium.
10
On the other hand, Wallace and Miller
stated that NO plays a critical role in modulating several
components of mucosal defence, including increased gastric
blood flow, reduced neutrophil adhesion, and increased
mucus secretion.
9
However, the relationship between iNOS
and NSAID induced gastric damage has not been studied
extensively and requires further clarification.
The recent development of genetically engineered mice
with gene deletions (knockout) makes it theoretically
possible to identify the specific inflammatory mediators that
may be involved in NSAID induced gastropathy.
6
In the
present study, we optimised a C57BL/6 mouse model of
indomethacin induced gastropathy to evaluate the role of
TNF-aand iNOS generated NO in gastric damage and
granulocyte infiltration using TNF-R1 and iNOS deficient
mice.
MATERIAL AND METHODS
Animals
C57BL/6 mice (weight 18–20 g) were fasted for 18–24 hours
before the experiments. Breeding pairs of mice with a
targeted disruption of the TNF-areceptor R1 (p55) and
iNOS genes were obtained from Jackson Laboratories (Bar
Abbreviations: NSAIDs, non-steroidal anti-inflammatory drugs; NO,
nitric oxide; TNF-a, tumour necrosis factor a; TNF-R1, tumour necrosis
factor receptor 1; TNF-R2, tumour necrosis factor receptor 2; NOS, nitric
oxide synthase; iNOS, inducible nitric oxide synthase; MPO,
myeloperoxidase; TNF-R1 2/2, TNF-R1 deficient mice; iNOS 2/2,
iNOS deficient mice
791
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Harbor, Maine, USA). Breeding stock backcrossed to C57BL/6
was obtained and the genotype of p55 and iNOS mice was
determined by polymerase chain reaction of DNA, as
previously described.
11
Animals were housed in a sterile
laminar flow cabinet until the start of the experiments, in
temperature controlled rooms, and received water and food
ad libitum. All animal treatments and surgical procedures
were performed in accordance with the Guide for Care and
Use of Laboratory Animals, National Institutes of Health
(Bethesda, Maryland, USA).
Drugs
Drugs used in this study were indomethacin (Prodome
Quı´mica e Farmaceˆutica, Sa˜o Paulo, Sa˜o Paulo, Brazil), Tris
(Merck, Sa˜o Paulo, Sa˜o Paulo, Brazil), 1400W (Cayman
Chemical Co, Ann Arbor, Michigan, USA), L-NAME, thali-
domide, dexamethasone, fucoidin, 3,39, 5,59’-tetramethyl-
benzidine and hexadecyltrimethyl-ammonium bromide (all
from Sigma Chemicals, St Louis, Missouri, USA).
Gastric damage induce by indomethacin in mice
Gastric damage was induced in wild-type C57BL/6 mice by
intragastric installation of indomethacin (2.5, 5, 10, 20 mg/
kg) dissolved in Tris buffer. The control group received only
vehicle (Tris buffer). Animals were killed 6, 12 or 24 hours
later by decapitation after light anaesthesia. Other groups of
wild-type mice were treated with fucoidin, a sulphated
fucosylated polysaccharide that binds to and blocks the
function of L- and P-selectins
12 13
(two doses of 25 mg/kg, five
minutes before and six hours after, intravenously) or saline,
and then indomethacin was administered (10 mg/kg).
Twelve hours later, animals were sacrificed and their
stomachs rapidly removed, opened by an incision along the
greater curvature, and pinned onto a wax platform.
Haemorrhagic or ulcerative lesions were counted and their
length measured with analogue callipers. A gastric damage
score (lesion index) was then calculated as the sum of the
lengths of all linear erosions,
14
which was measured by an
observer (HPL) who was unaware of the treatment given to
the animals. After scoring the damage, a sample of the corpus
region of each stomach was excised for measurement of
myeloperoxidase (MPO) activity.
15
MPO is an enzyme found
primarily in the azurophilic granules of neutrophils and
therefore has been used extensively as a biochemical marker
of granulocyte infiltration into various tissues, including the
gastrointestinal tract.
Role of TNF-ain indomethacin induced gastric
damage in mice
Gastric damage was induced in TNF-R1 deficient mice (TNF-
R12/2) or wild-type C57BL/6 mice by intragastric installa-
tion of indomethacin (10 mg/kg). Other groups of wild-type
mice were treated with the TNF-asynthesis inhibitor
thalidomide
16
(50 mg/kg, one hour before, and 25 mg/kg,
six h after, intraperitoneally), or with the glucocorticoid
dexamethasone (two doses of 1 mg/kg, one hour before and
six hours after, intraperitoneally). One hour later, indo-
methacin was administered (10 mg/kg). The control group
received only vehicle. After a further 12 hours, gastric
damage was determined as described above. Thereafter, full
0
5
10
15
20
Lesion index (mm)
0
4
6
2
MPO activity (U/mg)
2.5C5102.5C 5 10 20
Indomethacin (mg/kg) Indomethacin (mg/kg)
AB
*
*
*
0
5
10
15
Lesion index (mm)
0
4
6
2
MPO activity (U/mg)
6C12246C1224
Time after indomethacin (h) Time after indomethacin (h)
CD
*
*
*
*
*
Figure 1 Intragastric instillation of indomethacin induced both gastric damage and an increase in gastric myeloperoxidase (MPO) activity in C57BL/6
mice. Animals were treated with indomethacin (2.5–20 mg/kg), and gastric damage (A) and gastric MPO activity (B) were determined 12 hours later.
The control (C) group received only Tris buffer. Gastric lesions and gastric MPO activity were greatest at a dose of 10 mg/kg. The time course of
indomethacin induced gastropathy showed that both gastric damage (C) and gastric MPO activity (D) peaked at 12 hours. Results are expressed as
means (SEM) of at least five animals per group. *p,0.05 compared with the control group (ANOVA and Newman-Keuls test).
792 Souza, Paula Lemos, Oliveira, et al
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thickness pieces of the gastric corpus were weighed, frozen,
and stored at 270˚
C until assay of MPO activity.
Role of iNOS derived NO in indomethacin induced
gastric damage in mice
Gastric damage was induced in iNOS deficient (iNOS2/2)or
wild-type C57BL/6 mice by intragastric instillation of
indomethacin (10 mg/kg). Other groups of wild-type or
iNOS2/2mice were treated with L-NAME (100 mg/kg, one
hour before, and 50 mg/kg, six hours after indomethacin
administration, intraperitoneally), and then indomethacin
was administered (10 mg/kg). Another group of wild type
mice was treated with a selective inhibitor of inducible NOS,
1400W
17
(1.5 mg/kg, one hour before, intravenously), and
then mice received indomethacin (10 mg/kg). The control
group received only vehicle. Twelve hours later, gastric
damage was determined as described above. Finally, full
thickness pieces of the gastric corpus were weighed, frozen,
and stored at 270˚
C until assay of MPO activity.
Gastric MPO activity
The extent of granulocyte accumulation in the gastric mucosa
was measured by assaying MPO activity, as previously
described.
15
Briefly, 50–100 mg of gastric tissue were homo-
genised in two volumes of ice cold buffer (0.1 M NaCl,
20 mM NaPO
4
, 15 mM Na EDTA), pH 4.7, and centrifuged at
3000 rpm for 15 minutes. The pellet was then subjected to
hypotonic lysis (900 ml of 0.2% NaCl solution followed
30 seconds later by addition of an equal volume of a solution
containing 1.6% NaCl and 5% glucose). After further
centrifugation, the pellet was resuspended in 50 mM
NaPO
4
buffer, pH 5.4, containing 0.5% H-TAB, and re-
homogenised. The homogenate was then frozen and thawed
three times and centrifuged again at 10 000 rpm for
15 minutes at 4˚
C. MPO activity in the resuspended pellet
was assayed by measuring the change in absorbance at
450 nm using tetramethylbenzidine (1.6 mM) and H
2
O
2
(0.5 mM). Firstly, the results were reported as total number
of neutrophils by comparing absorbance of the tissue
supernatant with that of rat peritoneal neutrophils processed
in the same way. To this end, neutrophil migration was
induced in the peritoneum of rats by injecting carrageenin
(300 mg/animal). A standard curve relating neutrophil
numbers (.90% purity, 12 500 to 195.3 neutrophils/50 ml)
and absorbance was obtained by processing purified neu-
trophils, as described above, and assaying for MPO activity.
The correlation between the number of neutrophils and units
of MPO was determined using the technique described by
Bradley and colleagues.
18
The neutrophil standard curve was
processed using 0.0005% hydrogen peroxide as substrate for
MPO. A unit of MPO activity was defined as that converting
1mmol of hydrogen peroxide to water in one minute at 22˚
C.
Statistical analysis
Statistical analysis was performed using one way analysis of
variance (ANOVA) followed by the Newman-Keuls test,
when appropriate. Statistical significance was set at p,0.05.
RESULTS
Intragastric administration of indomethacin induced linear
haemorrhagic erosions in the corpus of the animals’
stomachs. As shown in fig 1, indomethacin induced gastric
lesions and increased gastric MPO activity dose dependently,
with the greatest effect at a dose of 10 mg/kg (fig 1A, B). The
time course of both gastric damage and the increase in gastric
MPO activity induced by indomethacin, at a dose of 10 mg/
kg, peaked at 12 hours (fig 1C, D).
Gastric damage (fig 2A) and gastric MPO activity (fig 2B)
induced by indomethacin (10 mg/kg) were significantly
reduced by treatment with fucoidin. Figure 3 shows that
thalidomide or dexamethasone treatment significantly inhib-
ited both the gastric damage (fig 3A) and gastric MPO
activity (fig 3B) induced by indomethacin. Furthermore,
TNF-R1 deficient mice presented less gastric damage (fig 3C)
and gastric MPO activity (fig 3D) than wild-type mice.
Treatment with L-NAME or 1400W reduced the gastric
damage (fig 4A) induced by indomethacin. Gastric MPO
activity induced by indomethacin was increased with
L-NAME treatment and unchanged with 1400W treatment
(fig 4B). In addition, after indomethacin administration,
iNOS deficient mice had less severe gastric damage and
similar indomethacin induced gastric MPO activity compared
with wild-type mice (fig 4C, D).
Table 1 shows that L-NAME treatment did not change
indomethacin induced gastric damage in iNOS deficient mice
but increased gastric MPO activity compared with iNOS
deficient mice.
DISCUSSION
The recent development of genetically engineered mice
makes this species particularly attractive as an animal model
to define the pathogenic mechanism responsible for NSAID
induced gastric damage.
6
In the present study, we optimised
a mouse model of indomethacin induced gastropathy and
used TNF-R1 or iNOS deficient mice to evaluate the role of
TNF-aand iNOS derived NO in the gastric damage and
granulocyte infiltration induced by indomethacin. This last
event was analysed because indomethacin induces gastric
0
5
10
15
Lesion index (mm)
*
SalineC Fucoidin
Indomethacin (10 mg/kg)
0
2
4
6
8
MPO activity (U/mg)
*
SalineC Fucoidin
Indomethacin (10 mg/kg)
A
B
Figure 2 Gastric damage and gastric myeloperoxidase (MPO) activity
induced by indomethacin were significantly reduced by treatment with
fucoidin. Wild-type mice were treated with saline or fucoidin. Five
minutes later, indomethacin (10 mg/kg) was administered. Gastric
lesions (A) and gastric MPO activity (B) were determined 12 hours later.
The control (C) group received only Tris buffer. Results are expressed as
means (SEM) of at least five animals per group. *p,0.05 compared with
the saline group (ANOVA and Newman-Keuls test).
Indomethacin induced gastric damage in TNF-R1 or iNOS deficient mice 793
www.gutjnl.com
damage is a neutrophil dependent process.
2–6
Our data
showed that TNF-a, acting via TNF-R1, mediates gastric
damage and granulocyte infiltration, measured as MPO
activity, in indomethacin induced gastropathy.
Furthermore, iNOS generated NO is involved in gastric
damage induced by indomethacin.
Indomethacin induced dose and time dependent gastric
erosions and an increase in gastric MPO activity in mice, with
the greatest effect 12 hours after administration. Gastric
damage observed was associated with increased MPO activity
because the severity and time course of the gastric damage
were coincident with such infiltration. Moreover, treatment
with fucoidin, a sulphated fucosylated polysaccharide that
binds to and blocks the function of L- and P-selectins,
12 13
reduced both gastric MPO activity and gastric damage
induced by indomethacin. Similarly, other studies have
shown that NSAID induced gastric damage in rats depends
on neutrophil mucosal infiltration.
25
Furthermore, treatment
with anti- ICAM-1, P-selectin, and E-selectin attenuated the
severity of indomethacin induced gastric in rats,
19
and mice
deficient in L- or P-selectins developed less indomethacin
induced gastric damage.
6
We observed that TNF-a, acting via TNF-R1, is an
important mediator in NSAID induced gastric damage and
granulocyte infiltration as both events were reduced in
TNF-R12/2mice. The involvement of TNF-ain this event
was further supported by the observation that thalidomide, a
drug that inhibits TNF-aproduction,
16
and also dexametha-
sone, reduced both gastric MPO activity and gastric damage
induced by indomethacin. These results are in agreement
with previous studies that showed a reduction in gastric
susceptibility to indomethacin in rats treated with pentoxifyl-
line, thalidomide, or dexamethasone.
45
With regard to the
TNF-R1 receptor, there is much evidence that it is the main
receptor that mediates the inflammatory effect of TNF-a,
being responsible for neutrophil migration and activation,
including NO production.
7
Furthermore, nuclear factor kb,an
important transcription factor for inflammatory events,
including gastrointestinal inflammatory diseases,
20
is acti-
vated by TNF-avia the TNF-R1 receptor.
7
The glucocorticoid
dexamethasone, apart from inhibiting TNF-aproduction,
also inhibits release of the majority of other inflammatory
cytokines, such as interleukin1band chemokines,
21
and
lipoxygenase products,
21
which are mediators thought to be
involved in the gastrointestinal inflammatory process.
22
Our
results do not eliminate the possibility that these molecules
may also mediate indomethacin induced gastropathy in mice.
In spite of the many studies showing involvement of NO in
the gastric damage induced by chemical substances, such as
serotonin or compound 48/80,
23 24
by stress,
25
or by Helicobacter
pylori infection,
26
the role of iNOS derived NO in NSAID
induced gastric damage has not been completely elucidated.
There is evidence that low doses of NO releasing substances
protect against NSAID induced gastropathy
27
and increase the
healing rate of gastric ulcers.
28
However, high doses of these
substances could induce extensive haemorrhagic mucosal
damage.
29 30
Furthermore, there are studies showing that
L-NAME, a non-specific NOS inhibitor, at doses of 15–50 mg/kg,
0
5
10
15
Lesion index (mm)
0
4
6
2
MPO activity (U/mg)
SalineC Thalid DexaSalineC Thalid Dexa
Indomethacin (10 mg/kg) Indomethacin (10 mg/kg)
AB
*
*
*
*
0
10
20
30
40
Lesion index (mm)
0.0
1.0
1.5
0.5
MPO activity (U/mg)
WildC TNF-RI _/_
WildC TNF-RI _/_
Indomethacin (10 mg/kg) Indomethacin (10 mg/kg)
CD
*
*
Figure 3 Tumour necrosis factor a(TNF-a) mediates indomethacin induced gastric damage and an increase in gastric myeloperoxidase (MPO)
activity. Gastric damage (A) and gastric MPO activity (B) induced by indomethacin (10 mg/kg) were significantly reduced by treatment with
thalidomide (Thalid) or dexamethasone (Dexa), but not with saline. In addition, intragastric administration of indomethacin induced less gastric
damage (C) and gastric MPO activity (D) in tumour necrosis factor receptor 1 deficient mice (TNF-R12/2) than in wild-type mice (Wild). Results are
expressed as means (SEM) of at least five animals per group. *p,0.05 compared with the saline group (A, B) or wild-type mice (C, D) (ANOVA and
Newman-Keuls test).
794 Souza, Paula Lemos, Oliveira, et al
www.gutjnl.com
increases indomethacin induced gastric damage,
31
reduces
gastric blood flow,
32
and delays healing of chronic gastric
ulcers.
33
Our results showed that iNOS generated NO is
implicated in indomethacin induced gastric damage as iNOS
deficient mice exhibited less gastric damage when indo-
methacin was administered, and wild-type mice treated with
L-NAME at a dose of 100 mg/kg, which inhibits both cNOS
and iNOS activity,
34
or with 1400W, a selective inhibitor of
iNOS,
17
decreased indomethacin induced gastric damage. The
fact that both genetic (knockout mice) and pharmacological
(1400W) specific inhibition of iNOS reduced gastric damage
indicates that the effect of L-NAME on indomethacin induced
gastric damage was also determined by its effect on iNOS
activity, and not by causing a decrease in gastric blood flow,
which—due its inhibitory effect on cNOS—could, in turn,
render less conspicuous the haemorrhagic expression of the
gastric damage. Confirmation that the L-NAME effect was
due to inhibition of iNOS was demonstration that L-NAME
treatment did not enhance reduction of gastric damage
caused by indomethacin in iNOS deficient mice (table 1).
These results are in apparent contradiction with others
described in the literature which showed an increase in
indomethacin induced gastric damage by a non-specific NOS
inhibitor, L-NAME, at a dose of 50 mg/kg.
31
One possible
explanation for this discrepancy is that at this dose, L-NAME
is unable to efficiently inhibit iNOS activity.
With regard to the role of NO in granulocyte infiltration
induced by indomethacin, our results suggest that
small amounts of NO released by cNOS are sufficient to
downregulate indomethacin induced gastric granulocyte
0
10
20
30
Lesion index (mm)
0
4
6
8
2
0
4
6
8
2
MPO activity (U/mg)
SalineC L-NAME 1400WSalineC L-NAME 1400W
Indomethacin (10 mg/kg) Indomethacin (10 mg/kg)
AB
*
*
*
0
10
20
30
40
Lesion index (mm)
MPO activity (U/mg)
WildC iNOS _/_
WildC iNOS _/_
Indomethacin (10 mg/kg) Indomethacin (10 mg/kg)
CD
*
Figure 4 Involvement of inducible nitric oxide synthase (iNOS) generated nitric oxide in the gastric damage and increase in gastric myeloperoxidase
(MPO) activity by indomethacin. Treatment with L-NAME or 1400W reduced gastric damage (A) but promoted different effects on the indomethacin
induced increase in gastric MPO activity. L-NAME increased and 1400W did not change gastric MPO activity induced by indomethacin (B).
Furthermore, after indomethacin administration, iNOS deficient mice had less gastric damage than wild-type mice (Wild) (C), despite no change in
indomethacin induced gastric MPO activity (D). Results are expressed as means (SEM) of at least five animals per group. *p,0.05 compared with the
saline group (A, B) or wild-type mice (C, D) (ANOVA and Newman-Keuls test).
Table 1 Effect of L-NAME treatment in indomethacin induced gastropathy in iNOS
deficient mice
Group Lesion index*(mm) MPO activity*(U/mg)
Vehicle (Tris buffer) 0.0 (0.0) 0.64 (0.06)
Indomethacin (wild-type mice) 23.1 (3.7) 5.36 (0.26)
Indomethacin+saline (iNOS2/2) 12.5 (2.6) 6.46 (1.02)
Indomethacin+L-NAME (iNOS2/2) 9.3 (0.7)``` 8.52 (0.74)```
*Data are mean (SEM) of 4–6 mice per group.
iNOS, inducible nitric oxide synthase; MPO, myeloperoxidase.
p,0.01, indomethacin+saline (iNOS2/2) versus indomethacin (wild-type mice).
```p,0.001, indomethacin+L-NAME (iNOS2/2) versus indomethacin (wild-type mice).
Indomethacin induced gastric damage in TNF-R1 or iNOS deficient mice 795
www.gutjnl.com
infiltration. This interpretation is supported by the fact that
we observed enhancement of gastric MPO activity only when
both NOS isofoms (cNOS and iNOS) were inhibited by
L-NAME in both wild-type and iNOS2/2mice. In line with
our results, there is evidence in the literature that NO inhibits
expression of cell adhesion molecules on endothelial cells,
which is an important step in neutrophil migration.
35–37
The fact that the reduction in indomethacin induced
gastric lesions observed in iNOS2/2,inL-NAME or 1400W
treated mice, was not accompanied by a similar reduction in
granulocyte infiltration appears to contradict the hypothesis
that NSAID induced gastric damage is a neutrophil depen-
dent process. However, one possible explanation is that
although neutrophils, which could be the source of the NO
involved in gastric damage, are present in the gastric mucosa,
iNOS in these cells is not able to produce NO and/or other
reactive nitrogen species.
In summary, our results indicate that TNF-a, acting via
TNF-R1, is involved in indomethacin induced gastric damage
and neutrophil infiltration in mice. Furthermore, iNOS
generated NO is involved in gastric damage induced by
indomethacin.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the technical assistance of Ieda R
dos Santos Schivo. Grants from FAPESP, PRONEX, and CNPq
(Brazil) supported this work.
Authors’ affiliations
.....................
M H L P Souza, Department of Physiology and Pharmacology, School of
Medicine, Federal University of Ceara´, Brazil, and Department of
Medicine (Division of Gastroenterology), School of Medicine Ribeira˜o
Preto, University of Sa˜o Paulo, Brazil
H Paula Lemos, Department of Medicine (Division of Gastroenterology),
School of Medicine Ribeira˜o Preto, University of Sa˜o Paulo, Brazil, and
Department of Pharmacology, School of Medicine Ribeira˜o Preto,
University of Sa˜o Paulo, Brazil
R B Oliveira, Department of Medicine (Division of Gastroenterology),
School of Medicine Ribeira˜o Preto, University of Sa˜o Paulo, Brazil
F Q Cunha, Department of Pharmacology, School of Medicine Ribeira˜o
Preto, University of Sa˜o Paulo, Brazil
REFERENCES
1Laine L. Approaches to nonsteroidal anti-inflammatory drug use in the high-
risk patient. Gastroenterology 2001;120:594–606.
2Wallace JL, Keenan CM, Granger DN. Gastric ulceration induced by
nonsteroidal anti-inflammatory drugs is a neutrophil-dependent process.
Am J Physiol 1990;259:G462–7.
3Trevethick MA, Clayton NM, Strong P, et al. Do infiltrating neutrophils
contribute to the pathogenesis of indomethacin induced ulceration of the rat
gastric antrum?Gut 1993;34:156–60.
4Santucci L, Fiorucci S, Di Matteo FM, et al. Role of tumor necrosis factor alpha
release and leukocyte margination in indomethacin-induced gastric injury in
rats. Gastroenterology 1995;1108:393–401.
5Appleyard CB, McCafferty DM, Tigley AW, et al. Tumor necrosis factor
mediation of NSAID-induced gastric damage: role of leukocyte adherence.
Am J Physiol 1996;270:G42–8.
6Morise Z, Granger DN, Fuseler JW, et al. Indomethacin induced gastropathy
in CD18, intercellular adhesion molecule 1, or P-selectin deficient mice. Gut
1999;45:523–8.
7Tartaglia LA, Goeddel DV. Two TNF receptors. Immunol Today
1992;13:151–3.
8Muscara MN, Wallace JL. Nitric oxide. V. therapeutic potential of nitric oxide
donors and inhibitors. Am J Physiol 1999;276:G1313–16.
9Wallace JL, Miller MJ. Nitric oxide in mucosal defense: a little goes a long
way. Gastroenterology 2000;119:512–20.
10 Piotrowski J, Slomiany A, Slomiany BL. Activation of apoptotic caspase-3 and
nitric oxide synthase-2 in gastric mucosal injury induced by indomethacin.
Scand J Gastroenterol 1999;34:129–34.
11 Pfeffer K, Matsuyama T, Kundig TM, et al. Mice deficient for the 55 kd tumor
necrosis factor receptor are resistant to endotoxic shock, yet succumb to L
monocytogenes infection. Cell 1993;73:457–67.
12 Varki A. Selectin ligands. Proc Natl Acad Sci U S A 1994;91:7390–7.
13 Bevilacqua MP. Endothelial-leukocyte adhesion molecules. Annu Rev Immunol
1993;11:767–804.
14 Santucci L, Fiorucci S, Giansanti M, et al. Pentoxifylline prevents
indomethacin-induced acute gastric-mucosal damage in rats—role of tumor-
necrosis-factor-alpha. Gut 1994;35:909–15.
15 Souza DG, Cassali GD, Poole S, et al. Effects of inhibition of PDE4 and TNF-a
on local and remote injuries following ischaemia and reperfusion injury.
Br J Pharmacol 2001;134:985–94.
16 Sampaio EP, Sarno EN, Galilly R, et al. Thalidomide selectively inhibits tumor
necrosis factor alpha production by stimulated human monocytes. J Exp Med
1991;173:699–703.
17 Garvey EP, Oplinger JA, Furfine ES, et al. 1400W is a slow, tight binding,
and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in
vivo. J Biol Chem 1997;272:4959–63.
18 Bradley PP, Christensen RD, Rothstein G. Cellular and extracellular
myeloperoxidase in pyogenic inflammation. Blood 1982;3:618–22.
19 Wallace JL, McKnight W, Miyasaka M, et al. Role of endothelial adhesion
molecules in NSAID-induced gastric mucosal injury. Am J Physiol
1993;265:G993–8.
20 Schmid RM, Adler G. NF-kappaB/rel/IkappaB: implications in
gastrointestinal diseases. Gastroenterology 2000;118:1208–28.
21 Haynes RC. Adrenocorticotropic hormone; adrenocortical steroids and their
synthetic analogs; inhibitors of the synthesis and actions of adrenocortical
hormones. In: Gilman AG, Rall TW, Nies AS, et al, eds. The pharmacological
basis of therapeutics, 8th edn. New York: Pergamon Press, 1990:1431–62.
22 Wallace JL, Ma L. Inflammatory mediators in gastrointestinal defense and
injury. Exp Biol Med (Maywood) 2001;226:1003–15.
23 Yasuhiro T, Korolkiewicz RP, Kato S, et al. Role of nitric oxide in pathogenesis
of serotonin-induced gastric lesions in rats. Pharmacol Res 1997;36:333–8.
24 Yasuhiro T, Konaka A, Kato S, et al. Gastric mucosal damage induced by
compound 48/80: roles of serotonin and nitric oxide. J Gastroenterol Hepatol
1998;13:1099–106.
25 Nishida K, Ohta Y, Ishiguro I. Relation of inducible nitric oxide synthase
activity to lipid peroxidation and nonprotein sulfhydryl oxidation in the
development of stress-induced gastric mucosal lesions in rats. Nitric Oxide
1998;2:215–23.
26 Mannick EE, Bravo LE, Zarama G, et al. Inducible nitric oxide synthase,
nitrotyrosine, and apoptosis in Helicobacter pylori gastritis: effect of antibiot ics
and antioxidants. Cancer Res 1996;56:3238–43.
27 Calatayud S, Sanz MJ, Canet A, et al. Mechanisms of gastroprotection by
transdermal nitroglycerin in the rat. Br J Pharmacol 1999;127:1111–18.
28 Elliott SN, McKnight W, Cirino G, et al. A nitric oxide-releasing nonsteroidal
anti-inflammatory drug accelerates gastric ulcer healing in rats.
Gastroenterology 1995;109:524–30.
29 Lamarque D, Whittle BJ. Role of oxygen-derived metabolites in the rat gastric
mucosal injury induced by nitric oxide donors. Eur J Pharmacol
1995;277:187–94.
30 Gurbuz V, Alican I, Berrak, et al. Role of nitric oxide in indomethacin-induced
gastric mucosal dysfunction in the rat. Exp Physiol 1999;84:319–32.
31 Khattab MM, Gad MZ, Abdallah D. Protective role of nitric oxide in
indomethacin-induced gastric ulceration by a mechanism independent of
gastric acid secretion. Pharmacol Res 2001;43:463–7.
32 Ferraz JG, Tigley A, Wallace JL. Paradoxical effects of L-arginine on gastric
mucosal integrity. Eur J Pharmacol 1994;260:107–11.
33 Ma L, Wallace JL. Endothelial nitric oxide synthase modulates gastric ulcer
healing in rats. Am J Physiol Gastrointest Liver Physiol 2000;279:G341–6.
34 Ferraz JG, Sharkey KA, Reuter BK, et al. Induction of cyclooxygenase 1 and 2
in the rat stomach during endotoxemia: role in resistance to damage.
Gastroenterology 1997;113:195–204.
35 Fox-Robichaud A, Payne D, Kubes P. Inhaled NO reaches distal vasculatures
to inhibit endothelium- but not leukocyte-dependent cell adhesion. Am J Physiol
1999;277:L1224–31.
36 Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator of
leukocyte adhesion. Proc Natl Acad Sci U S A 1991;88:4651–5.
37 Benjamim CF, Silva JS, Fortes ZB, et al. Inhibition of leukocyte rolling by nitric
oxide during sepsis leads to reduced migration of active microbicidal
neutrophils. Infect Immun 2002;70:3602–10.
796 Souza, Paula Lemos, Oliveira, et al
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