Histopathologic Characterization of Acute Gastritis and Duodenitis

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Histopathologic Characterization of Acute Gastritis and Duodenitis
SHORT NOTE
Histopathologic Characterization of Acute Gastritis
and Duodenitis Induced by Inoculation of Escherichia
coli O157 in Mice
Toshinobu Horii1 and Miya Kobayashi2
From the 1Department of Laboratory Medicine, Hamamatsu University School of Medicine, 1-20-1
Handa-yama, Hamamatsu 431-3192 and 2Department of Functional Histology, Nagoya University
Postgraduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
Correspondence to: Toshinobu Horii, M.D. & Ph.D., Department of Laboratory Medicine, Hamamatsu University
School of Medicine, 1-20-1 Handa-yama, Hamamatsu 431-3192, Japan. Tel.: + 81-53-435-2788; Fax: + 81-53435-2794; E-mail: [email protected]
Microbial Ecology in Health and Disease 2002; 14: 247–251
Objective: To examine the effect of intragastric inoculation of an Escherichia coli O157 strain, which does not have such pathogenic
factors as the stx, eae and EHEC-hlyA genes, on the induction of acute gastritis and duodenitis in BALB/c mice. Design: Time-dependent
histopathologic changes were characterized in stomach and duodenum mucoepithelium by light and transmission electron microscopy.
Results: The acute gastritis was characterized by inflammation and polymorphonuclear leukocyte infiltration that was followed by
lymphocyte infiltration of the lamina propria, mucoepithelial erosions, mucoepithelial cell necrosis and haemorrhage from 1.5 to 24 h
after inoculation. At 24 h, lymphocytes infiltrated the mucoepithelium. The acute duodenitis was characterized by polymorphonuclear
leukocytic and lymphocytic infiltration of the lamina propria and by vacuolar degeneration of columnar epithelial cells from 1.5 to 24
h after inoculation. Inflammatory cells, including eosinophils, infiltrated mainly the lamina propria from 1.5 to 6 h after inoculation in
both stomach and duodenum. Conclusions: our results showed that E. coli O157 inoculation icduced acute gastritis and duodenitis,
independent of such pathogenic factors as stx, eae and EHEC-hlyA. Key words: Escherichia coli O157, infection model, acute gastritis,
acute duodenitis, BALB/c mouse.
INTRODUCTION
An association is recognized between bacterial colonization and gastric lesions such as peptic ulcers and those seen
in cases of acute gastritis (1). Animal models have provided information with respect to host–pathogen interactions (2), and microorganisms such as Helicobacter pylori,
Helicobacter felis and Gastrospirillum spp. have been associated with acute gastritis and gastric ulceration in such
models (3–6). Lipopolysaccharide (LPS) is a component of
the outer membrane of Gram-negative bacteria. Endotoxin, which is composed of LPS released from the bacterial cell wall, is an important factor in sepsis related to
Gram-negative infection. H. pylori endotoxin, for example, can induce gastric mucosal lesions in mice (4, 6).
Escherichia coli endotoxin has biological activity that includes inhibition of gastric acid and histamine secretion in
gastric mucosa (7–9). Small doses of endotoxin are reported to protect the gastric mucosa against damage by
noxious substances (8, 10). However, with the exception of
some microorganisms such as Helicobacter spp., little is
known of the effects of Gram-negative bacteria themselves
on the gastric mucosa.
© Taylor & Francis 2002. ISSN 0891-060X
In the present study, we characterized time-dependent
histopathologic changes in mouse stomach and duodenum
after a single intragastric inoculation of E. coli O157,
which does not have such pathogenic factors as the stx,
eae and EHEC-hlyA genes.
MATERIAL AND METHODS
Bacterial strains, media and culture conditions
E. coli O157 HU16, which does not have such ptahogenic
factors as the stx, eae and EHEC-hlyA genes and was
resistant to at least two antibiotics, ampicillin and tetracycline, was isolated from a patient with diarrhea. The MICs
of ampicillin and tetracycline for this strain were \ 256
and 256 mg/ml, respectively. These antibiotic resistance was
used for viable counts of E. coli HU16 as markes. Bacteria
were stored at − 70°C in heart infusion broth (Nissui
Pharmaceutical, Tokyo, Japan) containing 20% glycerol.
Subsequently, bacteria were inoculated on heart infusion
agar (Nissui) plates and incubated at 37°C overnight for
use.
Microbial Ecology in Health and Disease
248
T. Horii and M. Kobayashi
Animals
Eight- to ten-week-old BALB/c mice (Japan SLC, Hamamatsu, Japan) were used in the experiments. Mice were fed
a commercial rodent diet (Japan SLC) and provided water
ad libitum. Animal experiments were performed according
to the ethical guidelines of the Institute for Laboratory
Animal Research, Nagoya University School of Medicine,
Nagoya, Japan.
Experimental design
Twelve mice were divided into two groups, for inoculation
with bacterial suspension or phosphate-buffered saline
(PBS). Mice were injected intragastrically via an 18-gauge
feeding tube with 0.1 ml bacterial suspension (c. 109 cfu/
ml) or PBS once after 1 day of fasting. Two mice from
each group were killed at 1.5, 6 and 24 h after inoculation.
Mice were anesthetised with nembutal (Dainippon Pharmaceutical, Osaka, Japan) and killed before collection of
the entire stomach and duodenum.
The stomach was opened through the longer curvature
with sterile surgical instruments. The fundus, pylorus and
duodenum were then used for bacterial culture and microscopic examination. The experiment was repeated twice.
Microbiology
Fundus, pylorus and duodenum specimens were trimmed
with a razor to 3 ×5 mm, rinsed with PBS and homogenized with PBS. The homogenates were diluted serially in
PBS, and 20 ml from each dilution was plated onto
Mueller-Hinton agar (Nippon Becton Dickinson, Tokyo,
Japan) supplemented with 64 mg/ml ampicillin, 64 mg/ml
tetracycline, 10 mg/ml vancomycin and 3 mg/ml amphotericin B. Colonies were counted after 24 h of incubation
at 37°C under aerobic conditions.
Japan) for 2 min. The sections were then examined under
a transmission electron microscope (Hitachi H-7100; Hitachi, Tokyo, Japan).
RESULTS AND DISCUSSION
Microbiology
E. coli O157 colonies were recovered from the pylorus and
duodenum over 24 h, whereas no colonies were recovered
from the fundus at 6 and 24 h after inoculation (Fig. 1). In
the uninfected group, no colonies were recovered before or
after inoculation of PBS onto plates containing antibiotics.
Light microscopy and ultramicroscopy showed neither colonization nor invasion of E. coli in the fundus, pylorus
and duodenum up to 24 h after inoculation. It was likely
that colonies recoverd were E. coli, which remained or
grew in the stomach tube or at the surface, since the strain
showed acid resistance (data not shown).
Microscopic histopathology findings
In the E. coli-infected group, there was histopathologic
evidence of acute gastritis with infiltration of polymorphonuclear leukocytes (PMNLs) into the lamina propria,
mucoepithelial erosions and mucoepithelial cell necrosis
from 1.5 h to 6 h after inoculation (data not shown). At 24
h, observations included infiltration of lymphocytes into
the lamina propria, extensive mucoepithelial cell necrosis,
Light microscopy
Tissue fragments of stomach squamous mucosa and pylorus, and of duodenum were fixed in Karnovsky’s fixative
(11, 12). The tissues were then rinsed with PBS and stained
with 1% osmium tetroxide in 0.1 M cacodylate buffer pH
7.4, at room temperature for 90 min. After rinsing with
PBS, the tissues were dehydrated for 15-min periods in
increasing concentrations of ethanol (70, 80, 90, 95 and
100%, v/v) and embedded in a Quetol mixture (Nissin EM,
Tokyo, Japan). Semithin sections approximately 1 mm in
thickness were stained with toluidine blue and analyzed
under a light microscope (BS2; Olympus, Tokyo, Japan).
Transmission electron microscopy
For transmission electron microscopy (TEM), sections
were cut with a diamond knife on a Porter-Blum MT6000
ultramicrotome (RMC, Tucson, AZ, USA) and stained
with 1% uranyl acetate for 10 min, followed by a lead
staining reagent (Katayama Chemical Industries, Osaka,
Fig. 1. Viable counts (cfu/3 ×5 mm tissue) of E. coli O157 HU16
recovered from fundus --; pylorus --; duodenum --. They
are mean values from two experiments.
E. coli O157 -induced acute gastritis in mice
and vasodilation and haemorrhage in the mucoepithelium
(data not shown). In the duodenum of E. coli-infected
mice, acute duodenitis was characterized by the infiltration
of lymphocytes and PMNLs into the lamina propria and
necrosis of the mucoepithelium from 1.5 to 24 h after
inoculation (data not shown). The number of lymphocytes
identified was greater than the number of PMNLs. In the
fundus, no histopathologic changes were detected. The
249
group injected with PBS showed no evidence of acute
mucosal lesioning.
Ultramicroscopic histopathology findings
At 1.5 h after inoculation, mucoepithelial erosions was
detected in the pylorus. After 6 h, eosinophils, neutrophils
and macrophages had infiltrated into the lamina propria of
the pylorus and edema had occured (Fig. 2A). Eosinophil
Fig. 2.
250
T. Horii and M. Kobayashi
Fig. 2. Transmission electron micrographs of mouse pylorus and duodenum
after a single intragastric inoculation of
E. coli O157 HU16; (A) after 6 h,
eosinophils and neutrophils have
infiltrated into the lamina propria of the
pylorus and edema has occured; (B) after
6h, platelets are increased in the blood
vessels of the lamina propria and in the
mucoepithelium of the pylorus; (C) after
6 h, mucous cells of the pylorus show
necrotic degeneration (arrow). The muscularis mucosa is preserved; (D) after 24
h, lymphocytes have infiltrated into the
lamina propria of the pylorus. Intercellular spaces are enlarged (*); (E) after 1.5
h, columnar epithelial cells of the duodenum show vacuolar degeneration; (F)
after 6 h, cells in the terminal regions of
duodenal glands show necrotic degeneration (arrow). Eosinophils infiltrated into
the lamina propria; (G) after 24 h, the
necrotic degeneration of cells in the terminal regions of duodenal glands is
severe. EN, endothelial cell; Eo,
eosinophil; L, lymphocyte; Mc, mucous
cell; M, muscular layer; N, neutrophil; P,
platelet; R, red blood cell. Bar=1 mm.
infiltration was most marked; lymphocyte infiltration was
absent. Platelets increased in the blood vessels of the
lamina propria and in the mucoepithelium (Fig. 2B). Chief
cells, parietal cells and mucous cells of the pylorus showed
degeneration or necrosis, but the muscularis mucosa was
preserved (Fig. 2C). At 24 h, lymphocytes had infiltrated
mainly into the lamina propria and mucoepithelium of the
pylorus (Fig. 2D). Intercellular spaces were enlarged (Fig.
2D). Mucoepithelial cells at the luminal surface were
severely degenerated, whereas cells at the basal regions
showed mild degeneration. Large amounts of debris were
present in the mucus.
In the duodenum, vacuolar degeneration of columnar
epithelial cells and inflammation with moderate infiltration
of lymphocytes but with a decreased number of PMNLs
were observed at 1.5 to 24 h after inoculation (Fig. 2E) (1).
The degeneration of basal cells was more severe than that
of cells at the luminal surface. At 1.5 h after inoculation,
lymphocytes and PMNLs such as eosinophils, neutrophils
and macrophages were found in the lamina propria. The
infiltration of eosinophils was predominant. The cells in
the terminal regions of duodenal glands were preserved. At
6 h after inoculation, cells in the terminal regions of
duodenal glands showed degeneration (Fig. 2F). Partial
erosions of columnar epithelium was prominent and blood
vessels were exposed to the lumen. Inflammation in the
lamina propria was severe, with infiltration of
lymphocytes, plasma cells and PMNLs. At 24 h after
inoculation, the degeneration of gland cells was severe;
normal columnar epithelial cells increased in number and
mast cells had infiltrated the columnar epithelium (Fig.
2G).
Our results at 24 h matched patterns of gastritis induced
by H. pylori in animals. Such gastritis tends to be
lymphocytic and to lack neutrophil infiltration (2). At
earlier time points, however, the infiltration of PMNLs
into the pylorus was followed by infiltration of lymophocytes. The infiltration of varying numbers of
E. coli O157 -induced acute gastritis in mice
eosinophils was found in the lamina propria, as is seen in
most types of gastritis (13).
Gastric mucosal lesions are important complications of
septic shock and endotoxemia (6). LPS is thought to act
as a pathogen in endotoxemia-induced gastric lesions (8).
E. coli endotoxin has biologic activity including inhibition
of gastric acid and histamine secretion in the gastric mucosa (7– 9). In the present study, the acute gastritis and
duodenitis were induced by E. coli O157 inoculation,
independent of such pathogenic factors as stx, eae and
EHEC-hlyA, and microscopies showed no invasion of E.
coli in the tissues examined. Our results suggest that these
lesions may be responsible for surface components of
bacteria, i. e. LPS. Although it is unclear whether bacterial LPS is necessary for this effect, it may play a role in
stimulation of cytokine release from inflammatory cells
(7).
Haemorrhage of the gastric mucosa was observed at 24
h after E. coli inoculation. At dissection, clots filling the
stomach lumen were identified clearly. The histopathologic findings showed the haemorrhaging resulted from
extensive necrosis as well as vasodilation in the mucoepthelium. This E. coli O157 model may be useful in
investigating the mechanisms of gastric mucosal haemorrhage associated with bacterial gastritis.
We showed that a single intragastric inoculation of E.
coli O157 could induce acute gastritis and duodenitis in
mice. This may be important ecologically since some
foods or drinks such as cow-related products are exposed
to contamination with E. coli O157. Our results suggest
that ingestion of foods or drinks contaminated with an
increased number of E. coli O157 may produce harmful
effects on stomach as well as intestine.
In conclusion, a single intragastric inoculation of E.
coli O157 induced acute gastritis and duodenitis in
BALB/c mice, independent of such pathogenic factors as
stx, eae and EHEC-hlyA. The acute gastritis was characterized by polymorphonuclear leukocytic inflammation
that was followed by lymphocytic inflammation in the
lamina propria, mucoepithelial erosions, mucoepithelial
cell necrosis and haemorrhage. The acute duodenitis was
characterized by polymorphonuclear leukocytic and
lymphocytic inflammation in the lamina propria as well
as vacuolar degeneration of columnar epithelial cells. Our
results showed that E. coli O157 bacteria themselves induce severe mucoepithelial lesions independent of effects
of Stx.
251
ACKNOWLEDGEMENTS
This work is supported by a Grant-in-Aid for Scientific Research
(13771452) from the Ministry of Education, Culture, Sports,
Science and Technology of Japan and a grant for scientific
research from the Uehara Memorial Foundation, Tokyo, Japan.
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