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Infection and Immunity, February 2000, p. 664-671, Vol. 68, No. 2
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cytokine Expression and Production by Purified Helicobacter
pylori Urease in Human Gastric Epithelial Cells
Toshihito
Tanahashi,1,*
Masakazu
Kita,2
Tadashi
Kodama,1
Yoshio
Yamaoka,1
Naoki
Sawai,1
Tomoyuki
Ohno,1
Shoji
Mitsufuji,1
Ya-Ping
Wei,2
Kei
Kashima,1 and
Jiro
Imanishi2
Third Department of Internal
Medicine,1 and Department of
Microbiology,2 Kyoto Prefectural University
of Medicine, Kyoto 602-8566, Japan
Received 14 April 1999/Returned for modification 16 June
1999/Accepted 11 November 1999
 |
ABSTRACT |
Cytokines have been proposed to play an important role in
Helicobacter pylori-associated gastroduodenal diseases, but
the exact mechanism of the cytokine induction remains unclear. H. pylori urease, a major component of the soluble proteins
extracted from bacterial cells, is considered to be one of the
virulence factors for the inflammation in the gastric mucosa that is
produced in H. pylori infection. However, the response of
human gastric epithelial cells to the stimulation of urease has not
been investigated. In the present study, we used human gastric
epithelial cells in a primary culture system and examined whether
H. pylori urease stimulates the gastric epithelial cells to
induce proinflammatory cytokines by reverse transcription-PCR and
enzyme-linked immunosorbent assay. First, by using peripheral blood
mononuclear cells (PBMC) and a gastric cancer cell line (MKN-45 cells),
we confirmed the ability of purified H. pylori urease to
induce the production of proinflammatory cytokines. Furthermore, we
demonstrated that the human gastric epithelial cells produced
interleukin-6 (IL-6) and tumor necrosis factor alpha, but not IL-8,
following stimulation with purified urease. The patterns of cytokine
induction differed among human PBMC, MKN-45 cells, and human gastric
epithelial cells. These results suggest that the human gastric
epithelial cells contribute to the induction of proinflammatory
cytokines by the stimulation of H. pylori urease,
indicating that the epithelial cells were involved in the mucosal
inflammation that accompanied H. pylori infection.
| |
INTRODUCTION |
Helicobacter pylori, a
microaerophilic gram-negative bacterium, has been identified as the
cause of chronic gastritis and peptic ulcer disease in humans
(18). H. pylori-associated gastroduodenal diseases are characterized by the severe infiltration of neutrophils, lymphocytes, monocytes, and plasma cells in the gastric mucosa. Indeed,
cure of the infection results in a notable reduction in these cells in
the gastric mucosa (3). Because the accumulation and
activation of inflammatory cells have been induced by the local
production of cytokines (16), cytokines are proposed to play
an important role in the pathogenesis of H. pylori-associated gastroduodenal diseases. Many investigators have
examined the relation between H. pylori infection and
mucosal cytokines (7, 14, 17, 19). We have studied the
cytokine expression patterns in gastric mucosal biopsy specimens by
using reverse transcription-PCR (RT-PCR) and have found that the levels
of interleukin-6 (IL-6), IL-7, IL-8, IL-10, and tumor necrosis factor
alpha (TNF-
) mRNA expression were significantly higher in H. pylori-positive than in H. pylori-negative patients
(29).
Several potential virulence factors derived from H. pylori
are considered to stimulate the cytokine induction in the gastric mucosa, thereby attracting and activating neutrophils and mononuclear cells (13, 21, 23). However, it remains unclear how these noninvasive bacteria, residing in the gastric mucous layer, produce the
inflammation and cause the damage to the underlying epithelial tissue.
Furthermore, the specific H. pylori protein responsible for
stimulating the cytokine induction has not been identified, although
the cag pathogenicity island was reported to be closely related to IL-8 production (5).
Urease plays a central role in the pathogenesis of H. pylori
infection by protecting the bacteria from the acid environment of the
stomach, promoting colonization, and inducing the production of
ammonia. In addition, recent studies showed that urease is a potent
chemoattractant factor for monocytes obtained from peripheral blood
mononuclear cells (PBMC) and mucosal macrophages (11, 12).
However, the response of the gastric epithelial cells to H. pylori urease has not been studied. The studies reported so far
focused on the results of in vitro culture using gastric cancer cell
lines (7, 13, 22).
In the present study, we established a primary culture system for human
gastric epithelial cells derived from the human stomach tissue at
surgery. To obtain insight into the mechanism of the cytokine
induction, we examined the response of the human gastric epithelial
cells to purified H. pylori urease.
| |
MATERIALS AND METHODS |
Preparation of purified H. pylori urease.
Purified H. pylori urease was provided by the Institute of
Immunology, Co., Ltd. (Tokyo, Japan) and prepared as described previously (9). In brief, for the urease purification,
H. pylori (ATCC 43504) was harvested from horse blood agar
plates and washed with phosphate-buffered saline (PBS) three times by
centrifugation at 5,000 × g for 10 min and resuspended
in PBS. Intact bacterial cells were ruptured by French pressure (20,000 lb/in2) cell lysis, and urease was purified from the
soluble protein by column chromatography on Sephacryl S-300HR
(Pharmacia, Inc., Piscataway, N.J.). When this material was
electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel (10%
acrylamide) and stained with Coomassie blue, only three bands
corresponding to the 62-, 60-, and 31-kDa proteins were detected,
indicating a purified enzyme preparation (Fig.
1). The specific identification of the
material was confirmed by Western blotting with antisera recognizing
the 31- and 62-kDa H. pylori urease (UreA and UreB, respectively) polypeptide, and the 60-kDa H. pylori heat
shock protein (hsp60) polypeptide. To prepare the specific material of
H. pylori UreB, the 62-kDa protein was separated and
concentrated after a sodium dodecyl sulfate-polyacrylamide gel
electrophoresis. The homogeneity of H. pylori UreB was
verified by silver staining by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (Fig. 1). That there was no endotoxin in the
purified urease preparation was confirmed by the manufacturer
(detection limit, <0.03 ng/ml).
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FIG. 1.
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis profile of prepared protein. (A) Purified H. pylori urease stained with Coomassie blue. Lane a, purified
H. pylori urease; lane b, molecular masses (in kilodaltons).
(B) Highly purified urease, including only the UreB protein, after
silver staining. Lane a, purified UreB; lane b, molecular masses (in
kilodaltons).
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|
PBMC preparation and the cell line.
Heparinized venous blood
drawn from healthy adult volunteers was diluted in PBS. Mononuclear
cells were separated by Ficoll-Hypaque (Pharmacia BiotechAB, Uppsala,
Sweden) density gradient centrifugation as recommended by the
manufacturer. PBMC were cultured at a final concentration of
106 cells/ml in RPMI 1640 medium supplemented with 5%
fetal bovine serum (FBS) and 2 mM glutamine in 24-well plates at 37°C
in 5% CO2.
In another series of experiments, the human gastric cancer cell line,
MKN-45, established from a poorly differentiated adenocarcinoma
metastasized to liver and obtained from the Japanese Cancer Research
Resource Bank (Tsukuba, Japan), was used. MKN-45 cells were seeded
in
24-well plates at 10
5 cells/ml in RPMI 1640 medium
supplemented with 5% FBS, 2 mM glutamine,
and 30 mg of streptomycin
per ml at 37°C in 5% CO
2.
Culture of human primary gastric epithelial cells.
Human
gastric epithelial cells were isolated enzymatically from the adult
human stomach as described earlier (24, 25). A piece of
healthy gastric mucosa (3 cm2) was obtained from the normal
fundic gland area of the stomach at surgery. The patients underwent
total gastrectomy because of early gastric cancer on the antrum. The
patients gave informed consent, and this study was approved by the
Human Research Committee of Kyoto Prefectural University of Medicine,
Kyoto, Japan. The surface mucosal layer was carefully removed with a
razor blade and minced immediately. The minced tissue was incubated in
Ham's F-12 culture medium containing collagenase type I (0.2 mg/ml) (Gibco BRL, Gaithersburg, Md.) for 10 min. Cells from the final incubation were washed and cultured in Ham's F-12 medium supplemented with 10% FBS and 30 mg of streptomycin per ml at 37°C in a
humidified 5% CO2 atmosphere. Human gastric epithelial
cells in a 24-well collagen-coated dish were cultured at a final
concentration of 106 cells/ml for 24 h before the
experiment. When the stimulation studies were performed, F-12 medium
was not supplemented with the FBS. Cultured cells had formed
subconfluent monolayers within 24 h of the inoculation. For the
histochemical identification of cultured cells, periodic acid-Schiff
(PAS) reaction was employed after 24 h of incubation. The cultured
cells in the monolayers had PAS-positive material in the cytoplasm,
indicating that the population consisted of only mucus-producing
epithelial cells without contamination by other cells (Fig.
2). Each experiment used gastric cells
from a different patient. However, individual experiments were
performed by using gastric cells from a single patient. The results
were obtained from three different experiments.
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FIG. 2.
Identification of human gastric epithelial cells in
primary culture. (A) Phase-contrast micrograph of human gastric
epithelial cells in primary culture (magnification, ×40). (B) PAS
staining of human gastric epithelial cells (magnification, ×40). The
PAS-positive material in the cytoplasm can be seen; an example is
indicated by the arrow.
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Detection of cytokine mRNA expression by RT-PCR.
PBMC,
MKN-45 cells, and human gastric epithelial cells were incubated for
3 h with purified H. pylori urease (0.1, 1, or 10 µg/ml), and total cellular RNA was extracted by the acid guanidinium isothiocyanate-phenol-chloroform method as described elsewhere (29). Aliquots (1 µg) of total RNA were incubated at
65°C for 5 min, chilled on ice, and reverse-transcribed in a final
volume of 10 µl of a solution containing 50 mmol of Tris-HCl (pH
8.3), 75 mmol of KCl, 3 mmol of MgCl2, 10 mmol of
dithiothreitol, and 200 mmol each of dATP, dCTP, dGTP, and dTTP
(Pharmacia BiotechAB, Uppsala, Sweden) per liter, plus 1 mmol of
oligo(dT) 16 primer per liter, 20 U of RNase inhibitor (Toyobo Co.,
Ltd., Tokyo, Japan), and 100 U of Moloney murine leukemia virus reverse
transcriptase (Gibco BRL, Gaithersburg, Md.). The mixture was incubated
at 43°C for 90 min, heated to 90°C for 10 min, and stored at
20°C until used.
The cDNA (5 µl) were added to 50-µl reaction mixtures containing 5 µl of 10× PCR reaction buffer, which consisted of 100 nmol
of KCl,
20 nmol of Tris-HCl (pH 7.5), 15 nmol of MgCl
2, 1 nmol
of
dithiothreitol, and 0.1 nmol of EDTA per liter, 200 ml of each
deoxynucleotide (Pharmacia Biotech AB, Uppsala, Sweden), 200 nmol
of
each primer per liter, 1.0 U of the
Taq DNA polymerase
included
in the Expand High Fidelity PCR system (Boehringer GmbH,
Mannheim,
Germany), and H
2O. The PCR was performed with an
automatic thermal
cycler (Takara PCR Thermal Cycler TP-3000; Takara
Biomedicals,
Otsu, Japan). The amplification cycle consisted of an
initial
denaturation of target DNA at 95°C for 5 min and then
denaturation
at 94°C for 1 min, annealing at 60°C for 1 min, and an
extension
step at 72°C for 1 min. The final cycle included an
extension
step for 7 min at 72°C to ensure full extension of the
product.
Aliquots (10 µl) of each PCR product were analyzed by
electrophoresis
on 1.5% Agarose S (Wako Chemical Co., Ltd., Osaka,
Japan) gels
containing ethidium bromide, and the bands were examined
under
UV light for the presence of the amplified
DNA.
Oligonucleotide primers were designed based on the previous study and
recent reports (
10,
27,
29). The sense and antisense
primers
specific for each cytokine were designed to include at
least one
intron, permitting distinction between amplified cDNA
and possible
contaminating residual genomic DNA. Table
1 shows
cytokine-specific oligonucleotide
primers. The
-actin gene was
assayed as a positive control for
cytokine mRNA expression. Primer
sequences were as follows: forward,
5'-GTGGGGCGCCCCAGGCACCA-3';
reverse,
5'-CTCCTTAATGTCACGCACGATTTC-3'.
Cytokine protein measurement.
PBMC and MKN-45 cells were
incubated for 24 h with purified H. pylori urease (0.1, 1, or 10 µg/ml), and the supernatants were collected and stored at
80°C until assayed. In addition, PBMC and MKN-45 cells were
incubated for 24 h with purified H. pylori UreB (0.1, 1, or 10 µg/ml), and the supernatants were collected. Human gastric
epithelial cells were incubated for 24 h with purified H. pylori urease (10 µg/ml), and the supernatants were collected at
3, 6, 12, and 24 h after urease stimulation. In addition, the culture supernatants were collected at 12 h after the stimulation of purified H. pylori urease (0.1, 1, or 10 µg/ml).
Cytokine levels in the culture supernatants were determined by means of
a specific enzyme-linked immunosorbent assay (ELISA).
IL-6, IL-8, and
TNF-
in the culture supernatants were measured
by ELISA by using
commercially available assay kits (BioSource
International, Inc.).
Assays were performed in duplicate according
to the manufacturer's
instructions. In these assays, the lower
limits of detection were 2 pg/ml for IL-6, 10 pg/ml for IL-8,
and 1 pg/ml for TNF-
. Regarding
PBMC and MKN-45 cells, the same
experiments were repeated three times,
and the results from all
experiments were
used.
Statistical analysis.
All data in each experiment were
expressed as the mean ± 1 standard error (SE). The statistical
significance of the cytokine production response to urease protein was
evaluated by using Student's t test as previously indicated
(11). Differences between cytokine levels were considered
significant when P was <0.05.
| |
RESULTS |
Cytokine mRNA expression by H. pylori urease in PBMC
and MKN-45 cells.
To examine the ability of H. pylori
urease to induce the production of cytokines, the
cytokine-specific mRNA expressions in human PBMC and MKN-45 cells
were analyzed by RT-PCR.
In PBMC, IL-1
, IL-6, IL-8, IL-10, IL-12, IL-18, and TNF-
mRNAs
were constitutively detected before and after the stimulation
of the
purified urease. However, gamma interferon (IFN-
) mRNA
was detected
neither before nor after the stimulation of the purified
urease. The
expressions of IL-6, IL-8, and TNF-
mRNAs are shown
as
representative results in Fig.
3.
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FIG. 3.
Expressions of IL-6, IL-8, and TNF- mRNA in PBMC and
MKN-45 cells. The cells were exposed to purified H. pylori
urease, and RT-PCR was carried out as described in Materials and
Methods. These cells were exposed to medium alone (control) and
increasing concentrations of H. pylori urease for 3 h.
-Actin gene was assayed as a positive control. The results from PBMC
(A) and from MKN-45 cells (B) are shown.
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|
The expression of IL-6 and TNF-
mRNA was induced in response to
exposure to
H. pylori urease in MKN-45 cells. IL-1
, IL-8,
IL-12, and IL-18 mRNAs were constitutively detected both before
and
after the stimulation of the purified urease. IL-10 and IFN-
mRNAs
were detected neither before nor after the stimulation (Table
2 and Fig.
3).
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TABLE 2.
Cytokine mRNA expressions in MKN-45 cells and primary
gastric epithelial cells in response to the purified urease protein
examined by RT-PCRa
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Cytokine production by H. pylori urease and UreB in
PBMC and MKN-45 cells.
To examine the cytokine production after
the stimulation of H. pylori urease, human PBMC and MKN-45
cells were incubated with purified urease for 24 h, and the
culture supernatants were analyzed by ELISA. In addition, we assessed
the response of human PBMC and MKN-45 cells to stimulation with highly
purified H. pylori urease, including only the UreB protein.
The production of IL-6 and TNF-
, but not IL-8, increased
significantly in a dose-dependent manner after the addition of urease
to PBMC. In particular, a large amount of TNF-
was detected in
the
supernatants (Fig.
4). In MKN-45 cells,
the production of
IL-6, IL-8, and TNF-
was significantly induced by
the addition
of 10 µg of urease per ml. However, TNF-
production
was considerably
lower than that in the PBMC (Fig.
4). Similarly,
H. pylori UreB
protein stimulated the production of IL-6 and
TNF-
, but not IL-8,
significantly in a dose-dependent manner in
PBMC. The production
of IL-8 and TNF-
, but not IL-6, was also
significantly induced
by the addition of UreB protein in MKN-45 cells
(Fig.
5).
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FIG. 4.
Productions of IL-6, IL-8, and TNF- induced by
purified H. pylori urease. Cells were incubated in
triplicate, and the levels of each cytokine in the culture supernatants
with purified H. pylori urease (0.1, 1, or 10 µg/ml) and
medium alone (control) were assayed by ELISA as described in Materials
and Methods. The results from PBMC (A) and from MKN-45 cells (B) are
shown. The results are shown as the mean ± 1 SE. * and **,
statistically significant differences from the values for the
corresponding medium alone (control) (P < 0.05 and
P < 0.01, respectively).
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FIG. 5.
Productions of IL-6, IL-8, and TNF- induced by
purified H. pylori UreB. Cells were incubated in triplicate,
and the levels of each cytokine in the culture supernatants with
purified UreB (0.1, 1, 10 µg/ml) and medium alone (control) were
assayed by ELISA as described in Materials and Methods. The results
from PBMC (A) and from MKN-45 cells (B) are shown. The results are
shown as the mean ± 1 SE. * and **, statistically
significant differences from the values for the corresponding medium
alone (control) (P < 0.05 and P < 0.01, respectively).
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Although there was little difference in the pattern of cytokine
production between PBMC and MKN-45 cells, these results indicated
that
purified
H. pylori urease, especially the component of UreB,
could stimulate PBMC and MKN-45 cells to produce proinflammatory
cytokines.
Cytokine mRNA expression and production by purified H. pylori urease in human gastric epithelial cells.
To obtain
further insight into the mechanism of the cytokine induction, we
examined the effect of purified H. pylori urease in human
gastric epithelial cells in the primary culture system.
Human gastric epithelial cells were incubated with purified
H. pylori urease for 3 h, and the expressions of mRNAs were
analyzed
by RT-PCR. The expressions of IL-6 and TNF-
mRNAs were
induced
after the stimulation with the purified urease, and there was
no expression of mRNAs in the unstimulated cells. The expressions
of
IL-1
, IL-8, IL-10, and IL-18 mRNAs were constitutively observed
both
before and after the stimulation with the purified urease.
IL-12 and
IFN-
mRNA expressions, on the other hand, were detected
neither
before nor after the stimulation. There was a difference
in the pattern
of the cytokine expression between the cell line,
the MKN-45 cells, and
the primary gastric epithelial cells (Table
2 and Fig.
6).
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FIG. 6.
Expressions of cytokine-specific mRNA and productions of
IL-6, IL-8, and TNF- induced by purified H. pylori urease
in human gastric epithelial cells. (A) Human gastric epithelial cells
were exposed to medium alone (control) and increasing concentrations of
H. pylori urease for 3 h. The expressions of cytokine
mRNAs were analyzed by RT-PCR as described in Materials and Methods.
(B) Cells were incubated in triplicate for 12 h, and the levels of
each cytokine in the culture supernatants with medium alone (control)
and purified H. pylori urease (0.1, 1, 10 µg/ml) were
measured by ELISA as described in Materials and Methods. The results
are shown as the mean ± 1 SE. * and **, statistically
significant differences from the value of the corresponding medium
alone (control) (P < 0.05 and P < 0.01, respectively).
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We further studied the time kinetics of cytokine production in primary
gastric epithelial cells at various time intervals
(Fig.
7). When stimulated with purified urease
(10 µg/ml), epithelial
cells secreted IL-6 and TNF-
, but not IL-8,
in a time-dependent
manner. IL-6 production could be detected in the
culture supernatant
within 3 h after the addition of urease, and
the most active secretion
occurred at 12 h. IL-8 was
constitutively produced in agreement
with a previous finding
(
25), and there was no difference in
the production level
between control and stimulated cells. TNF-
production was detectable
at 3 h of exposure to urease and continued
for up to 24 h
after exposure, reaching a peak of 60 pg/ml.
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FIG. 7.
Time course of IL-6 (A), IL-8 (B), and TNF- (C)
production induced by purified H. pylori urease in human
gastric epithelial cells. Cells were incubated in triplicate, and the
levels of each cytokine in the culture supernatants with purified
H. pylori urease (10 µg/ml;  ) and medium alone
(control;  ) were measured by ELISA as described in Materials and
Methods. The error bars indicate the SE values from three
determinations.
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In a subsequent experiment, cytokine production was examined at 12 h after stimulation with various concentrations of purified
H. pylori urease (0.1, 1, or 10 µg/ml) (Fig.
6). The production
of
IL-6 and TNF-
, but not IL-8, was significantly induced by
the
addition of 10 µg of urease per ml compared to the level of
the
unstimulated cells. With this assay, IL-6 production was not
detected
upon the addition of 0.1 and 1 µg of urease per ml, although
IL-6
mRNA expression was observed after 3 h of
stimulation.
| |
DISCUSSION |
Several studies have indicated that infection with H. pylori induced the expression and production of various cytokines
in the gastric mucosa, and cytokines contribute to the pathogenesis of
H. pylori-associated gastroduodenal diseases (3, 7, 17, 19, 29). However, the contribution of the human gastric
epithelial cells to the cytokine induction has not been studied. To
examine the mechanism of the cytokine induction in the epithelial
cells, gastric cancer cell lines have been used in other investigations as an in vitro system (13, 23). However, this approach has limitations for examining the natural properties of cytokine induction because the cell lines were obtained from human cancer cells. Therefore, we have successfully established a primary culture system
and examined the property of cytokine induction in human gastric
epithelial cells.
It was proposed that the ability to stimulate cytokine induction is
associated with several gene products of H. pylori (13, 23). In spite of searches for the components of H. pylori related to the cytokine stimulation, the mechanism still
remains unclear, though the cag pathogenicity island was
found to be closely related to IL-8 induction (5, 26). Since
H. pylori exists in the gastric mucus layer overlying the
epithelium and does not invade the epithelial tissue, it is still
unclear how a noninvasive bacterium stimulates the induction of
proinflammatory cytokines in the gastric mucosa. H. pylori
urease, a component of soluble proteins released outside the bacterial
cells (20), plays a central role in the pathogenesis of
infection by promoting colonization and inducing the production of
ammonia. In addition, the present study indicates that urease may act
as a virulence factor by inducing the production of IL-6 and TNF- in
gastric epithelial cells. These results also extend other observations
that H. pylori urease induced the production of certain
cytokines in human peripheral blood monocytes and mucosal macrophages
(11, 12). Compared to the response of mucosal macrophages,
it is of interest to note that the level of TNF- production was
nearly the same but that the level of IL-6 production was decreased in
the epithelial cells. Despite differences in the amounts of cytokine
produced, the dose responses were similar regardless of the cell
origin. However, the time kinetic responses could not compared because
there were no data except ours.
IL-6 has broad biological effects on mononuclear cells, including
B-cell and T-cell differentiation and activation of macrophages (2). These biological properties may be relevant to the
pathogenesis of gastroduodenal inflammation. We have previously shown
that there were some factors other than the cag
pathogenicity island which induced IL-6 production in H. pylori infection (30). The present study showed that
IL-6 production was induced in response to urease in human gastric
epithelial cells. Meanwhile, immunohistochemistry revealed positive
staining for IL-6 in the epithelial cells localized to the superficial
and the neck regions of the stomach glands in H. pylori-infected patients (16). These results indicated that H. pylori urease contributes to IL-6 production, at
least in part, in human gastric epithelial cells, whereas ELISA did not
detect IL-6 production in low-dose urease stimulation that had been
judged positive by RT-PCR. The apparent contradiction of these data may
be explained by methodological problems. The RT-PCR system is usually
chosen to catch the minimal expression of mRNA because of its high
level of sensitivity and its reproducibility.
The stimulation of TNF- production was found in human PBMC, MKN-45
cells, and human gastric epithelial cells. TNF- was correlated with
the severity of infiltration of mononuclear and polymorphonuclear cells
in H. pylori-positive specimens (4, 30). In
addition, TNF- has been shown to stimulate the gastrin secretion in
vitro (28), suggesting a role for this cytokine in H. pylori-induced hypergastrinemia (15), which is linked
to increased gastric acid secretion. Based on these findings, we
speculate that certain bacterial components, including urease, are
related to the gastric acid secretion through the cytokine production.
However, at this point, further investigations using other techniques
are needed.
By H. pylori urease, IL-8 was induced only in MKN-45 cells,
not in human PBMC nor the human gastric epithelial cells, although the
site of IL-8 induction was considered to be the epithelial cells
(6). This discrepancy might be explained by the difference of cell characteristics between the cancer cell line and the primary culture cells. Furthermore, it was proposed that the receptors for
H. pylori in host cells or other components contributed to the differences between the cell lines and PBMC (1, 8). These results showed that H. pylori urease was unrelated to
the IL-8 production, providing indirect evidence that some other
H. pylori protein induces the IL-8 production in the
epithelial cells.
In conclusion, the present study is the first to indicate that the
human gastric epithelial cells were sensitive to H. pylori urease and participated in the production of proinflammatory cytokines, such as IL-6 and TNF-. These results suggest that the gastric epithelial cells play a role in the mucosal inflammation that accompanied H. pylori infection.
| |
ACKNOWLEDGMENTS |
We thank Hisakazu Yamagishi and Chohei Sakakura (First Department
of Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan) for
generously donating the surgical specimen of human stomach. We are also
grateful to Yukito Kawakami (Institute of Immunology, Co., Ltd., Tokyo,
Japan) for providing the purified H. pylori urease.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Third Department
of Internal Medicine, Kyoto Prefectural University of Medicine, 465 Kajiicho, Kawaramachi-Hirokoji Kamigyo-ku, Kyoto 602-8566, Japan. Phone: 81-75-251-5519. Fax: 81-75-251-0710. E-mail:
ttana{at}sun.kpu-m.ac.jp.
Editor:
J. R. McGhee
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Infection and Immunity, February 2000, p. 664-671, Vol. 68, No. 2
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
