Previous Article | Next Article 
Infection and Immunity, June 2001, p. 3800-3808, Vol. 69, No. 6
Department of Medical Microbiology and
Immunology, Göteborg University, Göteborg, Sweden
Received 2 November 2000/Returned for modification 9 February
2001/Accepted 5 March 2001
Helicobacter pylori infection can cause duodenal
ulcers and may also induce gastric adenocarcinoma. The bacteria
colonize the gastric mucosa and areas of gastric metaplasia in the
duodenum for decades, resulting in active chronic inflammation in the
infected areas. A characteristic feature of the infection is the
ongoing recruitment of neutrophils to the infected sites. To evaluate the role of H. pylori lipopolysaccharides (LPS) in the
recruitment of leukocytes to the gastric mucosa, we have examined the
cytokine and chemokine production from human monocytes stimulated with LPS isolated from different H. pylori strains, as well
as from several other gram-negative bacteria. Our results show that
H. pylori LPS induce a large production of
neutrophil-recruiting CXC chemokines (interleukin-8 and growth-related
oncogene alpha) from purified human monocytes, to almost the same
extent as Escherichia coli LPS. However, and in
agreement with previous studies, H. pylori LPS was
much less potent in inducing production of proinflammatory cytokines by
purified human monocytes and was also a weak inducer of the CC
chemokine RANTES. There was no difference between LPS preparations from
different H. pylori strains in their ability to induce
cytokines and chemokines. The preferential production of CXC chemokines
after stimulation with H. pylori LPS indicates an
important contribution of this molecule in maintaining neutrophil recruitment during the infection, irrespective of the infecting strain.
Infection with the gram-negative
bacterium Helicobacter pylori is associated with development
of gastric and duodenal ulcers and in some instances also with gastric
carcinoma (7). H. pylori infection is
characterized by an active chronic gastric inflammation with invasion
of polymorphonuclear as well as mononuclear cells. A local increase in
the expression of proinflammatory cytokines such as interleukin-1
(IL-1), IL-6, and tumor necrosis factor alpha (TNF- Infection with H. pylori does not always give rise to
gastric symptoms. Approximately 10 to 15% of infected individuals
develop dyspepsia or peptic ulcers, whereas the rest remain more or
less asymptomatic (39). The factors that determine the
outcome of an H. pylori infection are still relatively
unknown (43). There are indications that H. pylori strains differ in their ability to induce cytokine
production; e.g., an important feature of the bacteria for the clinical
outcome of the infection seems to be the presence of the
cytotoxin-associated gene A (cagA) and the cagA-associated ice gene, which are associated
with ulcer disease (18, 32, 41, 46). Furthermore, studies
from our laboratory and others have shown that other putative virulence
factors, e.g., expression of the adhesin BabA on certain Lewis
antigens, are associated with development of a symptomatic H. pylori infection (41, 46). However, host factors also
seem to be important for the clinical outcome, since studies with mice
and humans have shown different outcomes of the infection depending on
the genetic background of the host (13, 17, 34).
It is well known that bacterial lipopolysaccharides (LPS) may induce
both strong local and systemic inflammation in animals as well as
humans, and therefore, H. pylori LPS is one of the factors
that could potentially influence local gastric inflammation and the
clinical outcome during an H. pylori infection. In general, H. pylori LPS is much less potent in activation of
inflammatory cells than LPS from members of the family
Enterobacteriaceae, e.g., Escherichia coli and
Salmonella spp. (5, 24, 26). This may be
explained by the structural differences between the lipid A molecules
of LPS from H. pylori and the Enterobacteriaceae. Another unusual feature of H. pylori LPS is the presence of
Lewis blood group antigens on the carbohydrate side chains (2, 3, 10, 25, 26). In spite of its relatively low toxic activity, H. pylori LPS has been shown to activate inflammatory cells
to produce different cytokines and chemokines, such as TNF- Therefore, we have examined the cytokine and chemokine responses
induced by H. pylori LPS purified from bacteria isolated from asymptomatic carriers and duodenal ulcer patients, as well as LPS
preparations expressing different Lewis blood group antigens. These
responses were compared to those induced by LPS from several other
gram-negative species.
LPS preparations.
H. pylori bacteria from strains
Hel 73, Hel 230, Hel 234, and Hel 255 and Sydney strain 1 (SS1) were
cultured on horse blood agar plates. The different strains were
selected based on whether they were isolated from patients with
duodenal ulcers or from asymptomatic carriers as well as on the type of
Lewis antigens expressed on their LPS (Table
1). SS1 was originally provided by Adrian
Lee, Sydney, Australia (23) and had been adapted to growth
in mice by several passages. All the other strains had been isolated in
our laboratory from Swedish volunteers undergoing gastroscopy for
diagnostic purpose. After 3 days of culture on horse blood agar plates
in a microaerophilic milieu (i.e., 10% CO2, 5%
O2, and 85% N2) at 37°C,
the bacterial cells were harvested and LPS was prepared by the
hot-phenol-water method described by Westphal and Jann
(44), dialyzed, and freeze-dried. The final product was
analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
and silver staining and was shown to give a typical ladder formation
(42). The protein content was spectrophotometrically measured and shown to be less than 5% (wt/wt). In preliminary experiments LPS preparations that had been further purified by treatment with proteases, RNase, and DNase (42) gave rise
to cytokine responses identical to those of the more crude LPS
preparations (data not shown). Therefore, the crude LPS preparations
were used in the rest of the experiments. LPS isolated from the
bacteria Salmonella enterica serovar Newport,
Vibrio cholerae strain 569B, E. coli strain
H10407, and Haemophilus ducreyi strain 4747 (kindly provided
by T. Lagergård) and prepared according to the same procedure were
also included in the study.
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.6.3800-3808.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Helicobacter pylori
Lipopolysaccharides Preferentially Induce CXC Chemokine Production in
Human Monocytes
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
), as well as
high levels of the neutrophil-recruiting chemokine IL-8, in the
H. pylori-infected stomach has also been documented
(15, 30).
, IL-8, IL-1, and monocyte chemotactic protein-1 (5).
Nevertheless, the potential capacity of purified H. pylori
LPS to induce chemokine production has not been extensively studied.
Furthermore, it is not known whether LPS from different H. pylori strains may differ in their capacities to induce cytokine
and chemokine production.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Characteristics of the H. pylori strains used
Demonstration of Lewis blood group antigens on H. pylori. The H. pylori bacteria were characterized for Lewis blood group antigen (Ley, Lex, Lea, and Leb) expression in the LPS by use of a slightly modified whole-cell enzyme-linked immunosorbent assay (37, 41). Briefly, 100-µl portions of previously frozen bacteria diluted to an optical density (OD) of 0.2 at 600 nm, corresponding to 109 bacteria/ml, were incubated in 96-well Maxisorb (Nunc, Roskilde, Denmark) plates at 4°C overnight. After being blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), monoclonal antibodies (Signet Laboratory, Dedham, Mass.) against the different Lewis antigens diluted 1/1,500 in BSA-PBS-Tween were added to each well. After incubation for 90 min at 37°C, plates were developed with a horseradish peroxidase-conjugated goat antibody to mouse immunoglobulins (Jackson Immuno Research Laboratories Inc., West Grove, Pa.) followed by addition of H2O2 and o-phenylenediaminedihydrocloride (OPD) as a chromogen. The plates were read at 450 nm in a spectrophotometer (Multiscan MS; Labsystems, Helsinki, Finland), and a positive reaction was defined as an OD value above the mean OD value plus three standard deviations obtained for three Lewis antigen-negative samples included in the same test.
Volunteers and specimen collection. For the peripheral blood mononuclear cell (PBMC) studies, blood specimens from four healthy adult H. pylori-negative volunteers were used. For the subsequent experiments, in which isolated monocytes were used, buffy coats and corresponding sera were collected from healthy adult Swedish volunteers donating at the blood bank of the Sahlgrenska University Hospital, Göteborg, Sweden, on the day of the experiment. The volunteers providing blood and buffy coats gave informed consent to participate in the study, which was approved by the Ethical Committee at the Medical Faculty, Göteborg University. Serum antibody levels against H. pylori were analyzed as previously described (19). Of the 19 individuals from whom buffy coats were collected, 14 were H. pylori negative and 5 were H. pylori positive.
Isolation and fraction of PBMC. PBMC were isolated and separated on a standard Ficoll-Paque (Pharmacia, Uppsala, Sweden) gradient (11). The PBMC were washed twice in PBS with 1% human AB+ serum and either used undiluted, depleted of various cell subsets, or enriched for monocytes.
To deplete the PBMC suspension of different cell subsets, magnetic beads (Dynabeads; Dynal, Oslo, Norway) precoated with antibodies reacting with CD4, CD8, CD14, or CD19 were added to the PBMC suspension and the mixture was pelleted by centrifugation. After 45 min of incubation on ice the pellet was carefully resuspended, and after another 20 min the beads were removed by use of a magnet. The remaining cell suspension was collected in a new tube, and cells were counted and washed twice in PBS with 1% human AB+ serum. To isolate monocytes from PBMC, the percentage of monocytes in the PBMC suspension was determined by their light scatter characteristics in flow cytometry analysis of unstained cells. The PBMC were then resuspended in Iscove's medium containing 5% human AB+ serum, 1% L-glutamine, and 1% gentamycine (Iscove's complete medium) at a final concentration of 5 × 105 monocytes/ml. One hundred microliters of the suspension was incubated in 96-well plates for 2 h at 37°C in 5% CO2. After incubation, the wells were repeatedly washed with PBS with 1% human AB+ serum. The supernatants were collected and again analyzed by flow cytometry to confirm the adherence of the monocytes to the cell culture plate. To the remaining adherent monocytes 100 µl of Iscove's complete medium was added, and the plates were kept at 37°C in 5% CO2. The monocytes were either stimulated immediately or allowed to differentiate to macrophages by further incubation at 37°C in 5% CO2 for 1 week (20). Differentiation to macrophages was confirmed by analysis of myeloperoxidase content, which is lost during maturation from monocytes to macrophages (20). Macrophages and monocytes were lysed by use of 0.02% Triton X-100 in PBS, and myeloperoxidase was determined by addition of OPD (Sigma-Aldrich, Stockholm, Sweden) (20). Following 1 week of differentiation, the macrophages were stimulated with LPS, and at the same time the old medium was replaced with 100 µl of fresh Iscove's complete medium.Stimulation of PBMC subsets.
In initial experiments
unfractionated PBMC and cell suspensions depleted of different
lymphocyte subsets (2 × 105 cells) were
stimulated with LPS (10 µg/ml) from H. pylori Hel 73, E. coli, S. enterica serovar Newport, V. cholerae, and H. ducreyi for 24 h. Purified
monocytes and macrophages were stimulated with LPS (2.5 or 25 µg/ml)
from H. pylori SS1, Hel 73, Hel 230, Hel 234, and Hel 255, E. coli, S. enterica serovar Newport, V. cholerae, and H. ducreyi in triplicate and were
incubated for 48 h. In some experiments, the produced TNF- was
neutralized by adding a mouse monoclonal antibody to human TNF-
(Pharmingen, Becton Dickinson, Stockholm, Sweden) at an antibody
concentration of 2 µg/ml, a concentration that has previously been
shown to inhibit the effect of 100 ng of TNF- per ml on endothelial
cell production of IL-8. Nonstimulated PBMC, monocytes, or macrophages were used as controls. The supernatants were pooled, aliquoted, and
stored at 
80°C until cytokine analysis. All buffer and media used
in the experiment were tested and found to contain <0.03 endotoxin
unit of endotoxin per ml using the Limulus test.
Detection of cytokines and chemokines.
The levels of IL-8,
TNF-, IL-10, growth-related oncogene alpha (GRO-), RANTES, and
granulocyte-macrophage colony-stimulating factor (GM-CSF) in the
cell supernatants were determined using the enzyme-linked immunosorbent
assay technique. For the detection of IL-8, 96-well Maxisorb (Nunc)
plates were coated with mouse anti-human IL-8 (R&D Systems, Oxon,
United Kingdom) and incubated overnight at 4°C. The plates were
blocked with PBS containing 1% BSA (Sigma-Aldrich), and samples were
incubated at 4°C overnight. Biotinylated rabbit anti-human IL-8
antibodies (R&D Systems), followed by extravidin-horseradish peroxidase
(Sigma-Aldrich), were used for detection, employing hydrogen peroxide
and OPD as substrates. After addition of 25 µl of 1 M
H2SO4, the final reaction was measured in a spectrophotometer (Multiscan MS; Labsystems) at 492 nm. The same procedure was used when analyzing all cytokines and
chemokines. R&D Systems produced the GM-CSF, GRO-, and RANTES reagents. IL-10 and TNF- reagents were obtained from Pharmingen. Standard curves were constructed using recombinant cytokines, and the
detection limits were 15.6 pg/ml for IL-8; 31.2 pg/ml for GM-CSF,
RANTES, and IL-10; 78.1 pg/ml for TNF-; and 312 pg/ml for GRO-.
Statistical analysis. Differences were evaluated by use of one-way analysis of variance (ANOVA) (Kruskall-Wallis) followed by Dunn's multiple-comparison test.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Cytokine production by PBMC after LPS stimulation.
Stimulation of PBMC from four individuals with LPS (10 µg/ml) from
H. pylori Hel 73, E. coli H10407, S. enterica serovar Newport, V. cholerae 569B, and
H. ducreyi resulted in production of IL-8.
Stimulation with H. pylori LPS induced PBMC to produce IL-8
to the same extent as the other LPS preparations used (Fig. 1). In contrast, E. coli, and
S. enterica serovar Newport LPS induced 3.5 to 6 times more
IL-10 than H. pylori or V. cholerae LPS (Fig. 1).
Likewise, a relatively low production of TNF- was seen when PBMC
were stimulated with H. pylori LPS (Fig. 1) compared to the
other LPS preparations.
|
CD14+ cells are the main source of cytokines after
stimulation with H. pylori LPS.
In order to
determine which cell types in the PBMC suspension responded to H. pylori LPS, selected cell subsets were depleted from the PBMC
suspension with Dynabeads coated with different anti-CD antibodies and
the remaining cells were stimulated with LPS (10 µg/ml) from H. pylori Hel 73 or E. coli H10407. Depletion of the
CD14+ monocyte fraction from the PBMC suspension
resulted in a dramatic decrease in IL-8 and TNF- production after
stimulation with either LPS (Fig. 2). In
some cases, removal of CD4+ or
CD8+ T cells even increased the cytokine
production on a per-cell basis, which could depend on some regulatory
effect exerted by these cells. In this particular set of volunteers
H. pylori LPS induced even higher levels of IL-8 production
than E. coli LPS, while E. coli LPS was a more
potent inducer of IL-10 and TNF- production.
|
LPS from different H. pylori strains induce similar responses. Based on the depletion experiments, we used purified monocytes when comparing the effects of LPS isolated from different H. pylori strains. These were initiated with titration experiments to determine the optimal conditions for the LPS stimulation in our systems. We found that 25 µg of H. pylori LPS per ml yielded much higher cytokine responses than 2.5 µg of H. pylori LPS per ml. Cytokine levels were maximal after 48 h of LPS stimulation. Unstimulated monocytes were used as controls in all of the following experiments.
To determine if there were any differences between LPS preparations from different H. pylori strains, we compared cytokine responses after stimulation with LPS from H. pylori SS1, Hel 73, Hel 230, Hel 234, and Hel 255. Also included in these experiments were LPS from E. coli H10407, S. enterica serovar Newport, H. ducreyi, and V. cholerae 569B. Monocytes from five H. pylori-seronegative individuals were stimulated with the different LPS preparations at 25 or 2.5 µg/ml, and supernatants were collected after 48 h. In all instances 25 µg of H. pylori LPS per ml but not 2.5 µg of H. pylori LPS per ml stimulated monocytes to produce high levels of IL-8 (30 to 50 ng/ml) compared to that produced by the control (approximately 8 ng/ml). No differences in the IL-8 response to the different H. pylori LPS preparations could be detected. When we analyzed the content of IL-10 in the supernatants, only low, or sometimes even no, IL-10 production could be detected when monocytes were stimulated with any of the H. pylori LPS preparations. TNF- could be detected only when monocytes were stimulated with 25 µg of H. pylori LPS per ml, and the levels were always
lower than when they were stimulated with the other LPS preparations.
In contrast to H. pylori LPS, E. coli, S. enterica serovar Newport, V. cholerae, and H. ducreyi LPS were capable of stimulating monocytes to significant
production of all cytokines analyzed at both 2.5 and 25 µg of LPS per ml.
Similar levels of production by monocytes and macrophages after
stimulation with LPS.
Since the cells responding to LPS in the
gastric tissue are likely to be macrophages rather than monocytes, we
also stimulated macrophages which were differentiated in vitro from
monocytes from the same individuals as described above. Macrophages
were stimulated with LPS (25 µg/ml) from H. pylori Hel 73, Hel 230, and Hel 234, E. coli H10407, V. cholerae
569B, S. enterica serovar Newport, and
H. ducreyi. There were no major differences in the
distributions and levels of cytokine production between macrophages and
monocytes when production of IL-8, TNF-, and IL-10 was analyzed
(data not shown). Therefore, we used monocytes in the following experiments.
H. pylori LPS preferentially induces the CXC
chemokines GRO- and IL-8.
Based on these experiments, we
decided to use monocytes stimulated with LPS (25 µg/ml) from H. pylori Hel 73, Hel 230, and Hel 234 and E. coli H10407 for the subsequent experiments, in which we
analyzed production of GRO- and RANTES in addition to the previously
studied cytokines. Monocytes from a total of 19 individuals were
stimulated with LPS (25 µg/ml) from H. pylori Hel 73, H. pylori Hel 230, or E. coli H10407. Fifteen of
the 19 individuals were also stimulated with 25 µg of Hel 234 LPS per
ml. These LPS preparations were chosen since they express different
Lewis blood group antigens and originate from either asymptomatic
carriers or duodenal ulcer patients. H. pylori LPS induced
significantly increased levels of IL-8 and GRO- in almost all
individuals compared to levels induced by the unstimulated control.
(Fig. 3). The levels of RANTES, which
were already fairly high in unstimulated controls, were not
significantly increased compared to levels in the control after
stimulation with different H. pylori LPS (Fig. 3). In
addition, 25 µg of E. coli LPS per ml induced
significantly higher levels of IL-8, GRO-, and RANTES in almost all
individuals (Fig. 3) compared to levels in the unstimulated control.
However, there was no significant difference between IL-8 and GRO-
production from purified human monocytes stimulated with the different
H. pylori LPS preparations or with E. coli LPS.
|
and GM-CSF and the immunomodulatory cytokine IL-10 were
secreted from purified monocytes to a much lesser extent after
stimulation with H. pylori LPS than were the chemokines. Increased TNF- and GM-CSF levels could be detected in some
individuals when monocytes were stimulated with LPS from H. pylori Hel 73, Hel 230, and Hel 234 (Fig.
4), but the responses to H. pylori LPS were not significantly different from those of the
control cultures. In contrast, cells stimulated with E. coli
H10407 LPS secreted significantly increased levels of TNF- and
GM-CSF compared to levels secreted in both unstimulated cells and cells stimulated with H. pylori LPS. IL-10 could not be
detected in any of the cell cultures stimulated with H. pylori LPS but was seen in about half of the E. coli
LPS-stimulated cultures (Fig. 4). To determine if the LPS added really
induced the GM-CSF and IL-10, we did a blocking experiment by adding a
monoclonal antibody against TNF-, since TNF- can induce GM-CSF
and IL-10 production. Neutralization of TNF- did not influence
cytokine production induced by H. pylori LPS. On the other
hand, E. coli LPS-induced GM-CSF and IL-10 production was
partly blocked by addition of a monoclonal TNF- antibody (data not
shown). Finally, there were no major differences between H. pylori-infected and noninfected individuals in any of the
chemokines and cytokines analyzed.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
In this study we show that H. pylori LPS can induce
high levels of CXC chemokines in spite of its comparatively low
capacity to induce production of proinflammatory cytokines. In several previous studies H. pylori LPS has been shown to be less
potent in activating the immune system than LPS from other
gram-negative intestinal bacteria (i.e., Enterobacteriaceae)
(5, 16, 21, 24, 27, 33). These findings are confirmed by
the present study, which shows considerably lower levels of TNF-
after stimulation with H. pylori LPS than after stimulation
with E. coli LPS. Furthermore, we show that the
inflammation-downregulatory cytokine IL-10, as well as GM-CSF, is
produced to a much smaller extent after stimulation with H. pylori LPS than after stimulation with LPS isolated from other
bacterial species. Blocking experiments demonstrate that the IL-10 and
GM-CSF production resulting from stimulation with E. coli LPS was only partly caused by autocrine actions of TNF-. Therefore, the low ability of H. pylori LPS to induce these
cytokines reflects actual differences between the LPS preparations
examined and stands in contrast to the effects of LPS from all
other species used in this study. The low production of downregulatory
cytokines may perhaps also contribute to the continuous inflammation
seen during H. pylori infection.
H. pylori LPS binds to CD14 (21), like LPS from
other species, but many of the intracellular responses seen after
H. pylori stimulation, such as NF-
and protein kinase
C activation, are mediated by LPS-independent pathways (29,
40). The low level of inflammation induced by H. pylori LPS has even been speculated to be beneficial for bacterial
persistence (6, 8), since H. pylori infection
might not induce strong enough responses for the host to eradicate the bacteria.
The relatively low stimulatory activity of H. pylori LPS could be due to structural differences, such as different phosphorylation patterns and an unusual fatty acid composition of the lipid A part compared to those in LPS from other bacteria (16, 27, 28, 31, 38). Another potentially important feature is that different H. pylori strains modify their LPS by variable expression of the human Lewis blood group antigens on the oligosaccharide structure (1, 10, 25, 26). Indeed, certain Lewis antigens have been associated with duodenal ulcer disease (41, 46). There was, however, no difference in levels of cytokine or chemokine production after stimulation with LPS isolated from strains with different Lewis blood group antigens or from strains from patients with different symptoms, suggesting that it is the conserved lipid A portion or other regions of the LPS that are responsible for the activity. Studies using LPS from different strains of Salmonella spp. have suggested a possibility that rough strains have a greater capacity to induce inflammation than smoother strains. However, in preliminary studies we did not find any differences in the stimulatory activities of LPS prepared from the rough strain CCUG 17874 (42) and the other H. pylori LPS used (M. Innocenti, unpublished observations). Therefore, the individual outcomes of an H. pylori infection do not seem to be dependent on the LPS type expressed by the infecting strain. These results emphasize the importance of host factors in the development of H. pylori-induced gastric ulcers.
In sharp contrast to the low levels of cytokines induced by
stimulation with H. pylori LPS, the amounts of CXC
chemokines produced after stimulation were significantly increased.
Chemokines are subdivided into CC chemokine and CXC chemokine
families on the basis of the positions of their two N-terminal cysteine
residues. The CC chemokines (e.g., RANTES) are active on multiple
leukocyte subtypes, but they are generally inactive on granulocytes. In this study, we chose to analyze production of the CXC chemokines IL-8
and GRO-, which preferentially recruit neutrophils, as well as the
T-cell-chemotactic chemokine RANTES, which attracts both Th1 and Th2
subsets of T cells. When we analyzed supernatants from monocytes
stimulated with different H. pylori LPS, we found a
significant increase in CXC chemokine production but not in RANTES
production. More significant, however, is our observation that the
concentrations of IL-8 and GRO- induced by H. pylori LPS
were comparable to those induced by the generally more potent E. coli LPS. Our cell depletion experiments indicate that
monocytes/macrophages are among the major sources of IL-8 after
H. pylori LPS stimulation. Therefore, our results suggest
that H. pylori LPS is an important factor in the induction
of CXC chemokines and in H. pylori gastritis, and they could
partly explain the continuous infiltration of granulocytes to the site
of infection. The neutrophils probably also participate in further
recruitment of new inflammatory cells by their own production of IL-8
(4, 12) and secretion of digested bacterial metabolites
that are chemotactic for other granulocytes. In addition, TNF-,
which is produced during H. pylori infection, is a potent activator of endothelial cells and might therefore participate in the
recruitment of new inflammatory cells that can participate in the
eradication of the bacteria (9). The ability of H. pylori LPS to induce production of granulocyte-recruiting
chemokines could also be one mechanism behind the large recruitment of
inflammatory cells, especially neutrophils, to the infected metaplastic
areas in the duodenum (18). These cells could in turn be
responsible for some of the tissue damage and ulcer formation that are
suggested to originate at these sites.
Previous studies have shown that H. pylori infection induces
production of both neutrophil- and T-cell-recruiting chemokines in
gastric mucosa as well as in in vitro systems (15, 22, 35, 36,
45). In particular, a high level of IL-8 in the mucosa is a
characteristic finding in H. pylori gastritis (14, 15,
36). The role of LPS in chemokine production in vivo, however,
is hard to assess. In a recent study, Lindholm et al. used human
stomach explants stimulated with viable H. pylori bacteria and corresponding LPS to study acute chemokine production
(23a). As in our study, stimulation led to
significantly increased levels of GRO- and IL-8 that were almost as
high as those induced by whole bacteria. These findings also emphasize
the potential importance of H. pylori LPS for neutrophil
recruitment in H. pylori-associated gastritis.
In conclusion, we have shown that H. pylori LPS is
much less potent in inducing the production of proinflammatory
cytokines TNF-, IL-10, and GM-CSF by human monocytes and macrophages
than are LPS from several other species. In contrast, the CXC
chemokines IL-8 and GRO- were produced in large amounts in response
to H. pylori LPS, and this might be one of the factors
promoting the continuous influx of neutrophils into the H. pylori-infected gastric mucosa.
| |
ACKNOWLEDGMENTS |
|---|
We thank Ingela Ahlstedt for excellent technical assistance and Jan Konar (Blood Bank, Sahlgrenska University Hospital) for excellent help with recruitment of healthy volunteers.
This study was supported by grants from the Göteborg University Faculty of Medicine, the Astra Research Center (Boston, Mass.), the Swedish Medical Research Council (grant 16X-13055), Kungliga och Hvitfeldska Överskottsfonden, and Socialstyrelsen.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Dept. of Medical Microbiology and Immunology, Göteborg University, Guldhedsgatan 10 A, S-413 46 Göteborg, Sweden. Phone: 46 31 42 44 92. Fax: 46 31 82 01 60. E-mail: marianne.quiding{at}microbio.gu.se.
Editor: R. N. Moore
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. | Appelmelk, B. J., R. Negrini, A. P. Moran, and E. J. Kuipers. 1997. Molecular mimicry between Helicobacter pylori and the host. Trends Microbiol. 5:70-73[CrossRef][Medline]. |
| 2. |
Appelmelk, B. J.,
B. Shiberu,
C. Trinks,
N. Tapsi,
P. Y. Zheng,
T. Verboom,
J. Maaskant,
C. H. Hokke,
W. E. Schiphorst,
D. Blanchard,
I. M. Simoons-Smit,
D. H. van den Eijnden, and C. M. Vandenbroucke-Grauls.
1998.
Phase variation in Helicobacter pylori lipopolysaccharide.
Infect. Immun.
66:70-76 |
| 3. | Aspinall, G. O., and M. A. Monteiro. 1996. Lipopolysaccharides of Helicobacter pylori strains P466 and MO19: structures of the O antigen and core oligosaccharide regions. Biochemistry 35:2498-2504[CrossRef][Medline]. |
| 4. |
Bazzoni, F.,
M. A. Cassatella,
F. Rossi,
M. Ceska,
B. Dewald, and M. Baggiolini.
1991.
Phagocytosing neutrophils produce and release high amounts of the neutrophil-activating peptide 1/interleukin 8.
J. Exp. Med.
173:771-774 |
| 5. | Birkholz, S., U. Knipp, C. Nietzki, R. J. Adamek, and W. Opferkuch. 1993. Immunological activity of lipopolysaccharide of Helicobacter pylori on human peripheral mononuclear blood cells in comparison to lipopolysaccharides of other intestinal bacteria. FEMS Immunol. Med. Microbiol. 6:317-324[CrossRef][Medline]. |
| 6. | Blaser, M. J. 1992. Hypotheses on the pathogenesis and natural history of Helicobacter pylori-induced inflammation. Gastroenterology 102:720-727[Medline]. |
| 7. | Blaser, M. J. 1995. The role of Helicobacter pylori in gastritis and its progression to peptic ulcer disease. Aliment. Pharmacol. Ther. 9:27-30. |
| 8. | Blaser, J. M., and J. Parsonnet. 1994. Parasitism by the "slow" bacterium Helicobacter pylori leads to altered gastric homeostasis and neoplasia. J. Clin. Investig. 94:4-8. |
| 9. | Boehm, U., T. Klamp, M. Groot, and J. C. Howard. 1997. Cellular responses to interferon-gamma. Annu. Rev. Immunol. 15:749-795[CrossRef][Medline]. |
| 10. |
Boren, T.,
P. Falk,
K. A. Roth,
G. Larson, and S. Normark.
1993.
Attachment of Helicobacter pylori to human gastric epithelium mediated by blood group antigens.
Science
262:1892-1895 |
| 11. | Boyum, A. 1968. Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand. J. Clin. Lab. Investig. Suppl. 97:77-89[Medline]. |
| 12. | Cassatella, A. M., F. Bazzoni, M. Ceska, I. Ferro, M. Baggiolini, and G. Berton. 1992. IL-8 production by human polymorphonuclear leukocytes. The chemoattractant formyl-methionyl-leucyl-phenylalanine induces the gene expression and release of IL-8 through a pertussis toxin-sensitive pathway. J. Immunol. 148:3216-3220[Abstract]. |
| 13. | Cover, T. L. 1997. Helicobacter pylori transmission, host factors, and bacterial factors. Gastroenterology 113:S29-S30[CrossRef][Medline]. |
| 14. | Crabtree, J. 1996. Gastric mucosal inflammatory responses to Helicobacter pylori. Aliment. Pharmacol. Ther. 10:29-37. |
| 15. |
Crabtree, J.
1994.
Interlukin-8 expression in Helicobacter pylori infected, normal, and neoplastic gastroduodenal mucosa.
J. Clin. Pathol.
47:61-66 |
| 16. |
Geis, G.,
H. Leying,
S. Suerbaum, and W. Opferkuch.
1990.
Unusual fatty acid substitution in lipids and lipopolysaccharides of Helicobacter pylori.
J. Clin. Microbiol.
28:930-932 |
| 17. | Go, M. F. 1997. What are the host factors that place an individual at risk for Helicobacter pylori-associated disease? Gastroenterology 113:S15-S20[CrossRef][Medline]. |
| 18. | Hamlet, A., A. C. Thoreson, O. Nilsson, A. M. Svennerholm, and L. Olbe. 1999. Duodenal Helicobacter pylori infection differs in cagA genotype between asymptomatic subjects and patients with duodenal ulcers. Gastroenterology 116:259-268[CrossRef][Medline]. |
| 19. | Hamlet, A. K., K. I. Erlandsson, L. Olbe, A. M. Svennerholm, V. E. Backman, and A. B. Pettersson. 1995. A simple, rapid, and highly reliable capsule-based 14C urea breath test for diagnosis of Helicobacter pylori infection. Scand. J. Gastroenterol. 30:1058-1063[Medline]. |
| 20. | Johansson, A., and C. Dahlgren. 1992. Differentiation of human peripheral blood monocytes to macrophages is associated with changes in the cellular respiratory burst activity. Cell Biochem. Funct. 10:87-93[CrossRef][Medline]. |
| 21. | Kirkland, T., S. Viriyakosol, G. I. Perez-Perez, and M. J. Blaser. 1997. Helicobacter pylori lipopolysaccharide can activate 70Z/3 cells via CD14. Infect. Immun. 65:604-608[Abstract]. |
| 22. | Kusugami, K., T. Ando, M. Ohsuga, A. Imada, M. Shinoda, T. Konagaya, K. Ina, N. Kasuga, A. Fukatsu, S. Ichiyama, T. Nada, and M. Ohta. 1997. Mucosal chemokine activity in Helicobacter pylori infection. J. Clin. Gastroenterol. 25:S203-S210. |
| 23. | Lee, A. O. R., J. Ungria, Maria Corazon de Robertson, B. Daskalopuolos, and M. G. Dixon. 1997. A standardized mouse model of Helicobacter pylori infection: introducing the Sydney strain. Gastroenterology 112:1386-1397[CrossRef][Medline]. |
| 23a. | Lindholm, C., M. Quiding-Järbrink, H. Lönroth, and A.-M. Svennerholm. Induction of chemokine and cytokine responses by Helicobacter pylori in human stomach explants. Scand. J. Gastroenterol., in press. |
| 24. |
Mattsby-Baltzer, I.,
Z. Mielniczuk,
L. Larsson,
K. Lindgren, and S. Goodwin.
1992.
Lipid A in Helicobacter pylori.
Infect. Immun.
60:4383-4387 |
| 25. |
Monteiro, M. A.,
K. H. Chan,
D. A. Rasko,
D. E. Taylor,
P. Y. Zheng,
B. J. Appelmelk,
H. P. Wirth,
M. Yang,
M. J. Blaser,
S. O. Hynes,
A. P. Moran, and M. B. Perry.
1998.
Simultaneous expression of type 1 and type 2 Lewis blood group antigens by Helicobacter pylori lipopolysaccharides. Molecular mimicry between H. pylori lipopolysaccharides and human gastric epithelial cell surface glycoforms.
J. Biol. Chem.
273:11533-11543 |
| 26. | Moran, A. P. 1996. The role of lipopolysaccharide in Helicobacter pylori pathogenesis. Aliment. Pharmacol. Ther. 10(Suppl. 1):39-50. |
| 27. |
Moran, A. P.,
I. M. Helander, and T. U. Kosunen.
1992.
Compositional analysis of Helicobacter pylori rough-form lipopolysaccharides.
J. Bacteriol.
174:1370-1377 |
| 28. |
Moran, A. P.,
B. Lindner, and E. J. Walsh.
1997.
Structural characterization of the lipid A component of Helicobacter pylori rough- and smooth-form lipopolysaccharides.
J. Bacteriol.
179:6453-6463 |
| 29. | Munzenmaier, A., C. Lange, E. Glocker, A. Covacci, A. Moran, S. Bereswill, P. A. Baeuerle, M. Kist, and H. L. Pahl. 1997. A secreted/shed product of Helicobacter pylori activates transcription factor nuclear factor-kappa B. J. Immunol. 159:6140-6147[Abstract]. |
| 30. | Noach, L. A., N. B. Bosma, J. Jansen, F. J. Hoek, S. J. van Deventer, and G. N. Tytgat. 1994. Mucosal tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-8 production in patients with Helicobacter pylori infection. Scand. J. Gastroenterol. 29:425-429[Medline]. |
| 31. | Ogawa, T., Y. Suda, W. Kashihara, T. Hayashi, T. Shimoyama, S. Kusumoto, and T. Tamura. 1997. Immunobiological activities of chemically defined lipid A from Helicobacter pylori LPS in comparison with Porphyromonas gingivalis lipid A and Escherichia coli-type synthetic lipid A (compound 506). Vaccine 15:1598-1605[CrossRef][Medline]. |
| 32. | Peek, R. M., Jr., S. A. Thompson, J. P. Donahue, K. T. Tham, J. C. Atherton, M. J. Blaser, and G. G. Miller. 1998. Adherence to gastric epithelial cells induces expression of a Helicobacter pylori gene, iceA, that is associated with clinical outcome. Proc. Assoc. Am. Phys. 110:531-544[Medline]. |
| 33. | Perez-Perez, G. I., V. L. Shepherd, J. D. Morrow, and M. J. Blaser. 1995. Activation of human THP-1 cells and rat bone marrow-derived macrophages by Helicobacter pylori lipopolysaccharide. Infect. Immun. 63:1183-1187[Abstract]. |
| 34. |
Sakagami, T.,
M. Dixon,
J. O'Rourke,
R. Howlett,
F. Alderuccio,
J. Vella,
T. Shimoyama, and A. Lee.
1996.
Atrophic gastric changes in both Helicobacter felis and Helicobacter pylori infected mice are host dependent and separate from antral gastritis.
Gut
39:639-648 |
| 35. | Sharma, S. A., M. K. Tummuru, G. G. Miller, and M. J. Blaser. 1995. Interleukin-8 response of gastric epithelial cell lines to Helicobacter pylori stimulation in vitro. Infect. Immun. 63:1681-1687[Abstract]. |
| 36. | Shimoyama, T., and J. E. Crabtree. 1997. Mucosal chemokines in Helicobacter pylori infection. J. Physiol. Pharmacol. 48:315-323[Medline]. |
| 37. | Simoons-Smit, I. M., B. J. Appelmelk, T. Verboom, R. Negrini, J. L. Penner, G. O. Aspinall, A. P. Moran, S. F. Fei, B. S. Shi, W. Rudnica, A. Savio, and J. de Graaff. 1996. Typing of Helicobacter pylori with monoclonal antibodies against Lewis antigens in lipopolysaccharide. J. Clin. Microbiol. 34:2196-2200[Abstract]. |
| 38. |
Suda, Y.,
T. Ogawa,
W. Kashihara,
M. Oikawa,
T. Shimoyama,
T. Hayashi,
T. Tamura, and S. Kusumoto.
1997.
Chemical structure of lipid A from Helicobacter pylori strain 206 to 1 lipopolysaccharide.
J. Biochem.
121:1129-1133 |
| 39. | Telford, J. L., A. Covacci, R. Rappuoli, and P. Chiara. 1997. Immunobiology of Helicobacter pylori infection. Curr. Opin. Immunol. 9:498-503[CrossRef][Medline]. |
| 40. |
Terres, A. M.,
J. M. Pajares,
A. M. Hopkins,
A. Murphy,
A. Moran,
A. W. Baird, and D. Kelleher.
1998.
Helicobacter pylori disrupts epithelial barrier function in a process inhibited by protein kinase C activators.
Infect. Immun.
66:2943-2950 |
| 41. |
Thoreson, A. C.,
A. Hamlet,
J. Celik,
M. Bystrom,
S. Nystrom,
L. Olbe, and A. M. Svennerholm.
2000.
Differences in surface-exposed antigen expression between Helicobacter pylori strains isolated from duodenal ulcer patients and from asymptomatic subjects.
J. Clin. Microbiol.
38:3436-3441 |
| 42. | Tinnert, A., A. Mattsson, I. Bolin, J. Dalenback, A. Hamlet, and A. M. Svennerholm. 1997. Local and systemic immune responses in humans against Helicobacter pylori antigens from homologous and heterologous strains. Microb. Pathog. 23:285-296[CrossRef][Medline]. |
| 43. | van Doorn, L. J., C. Figueiredo, R. Sanna, A. Plaisier, P. Schneeberger, W. de Boer, and W. Quint. 1998. Clinical relevance of the cagA, vacA, and iceA status of Helicobacter pylori. Gastroenterology 115:58-66[CrossRef][Medline]. |
| 44. | Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharide: extraction with phenol-water and further applications of the procedure, p. 83-92. In R. Whitler (ed.), Methods in carbohydrate chemistry. Academic Press, New York, N.Y. |
| 45. |
Yamaoka, Y.,
M. Kita,
T. Kodama,
N. Sawai,
T. Tanahashi,
K. Kashima, and J. Imanishi.
1998.
Chemokines in the gastric mucosa in Helicobacter pylori infection.
Gut
42:609-617 |
| 46. |
Zheng, P. Y.,
J. Hua,
K. G. Yeoh, and B. Ho.
2000.
Association of peptic ulcer with increased expression of Lewis antigens but not cagA, iceA, and vacA in Helicobacter pylori isolates in an Asian population.
Gut
47:18-22 |
Infection and Immunity, June 2001, p. 3800-3808, Vol. 69, No. 6
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.6.3800-3808.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Basak, C., Pathak, S. K., Bhattacharyya, A., Mandal, D., Pathak, S., Kundu, M.
(2005). NF-{kappa}B- and C/EBP{beta}-driven Interleukin-1{beta} Gene Expression and PAK1-mediated Caspase-1 Activation Play Essential Roles in Interleukin-1{beta} Release from Helicobacter pylori Lipopolysaccharide-stimulated Macrophages. J. Biol. Chem.
280: 4279-4288
[Abstract] [Full Text] -
Enarsson, K., Brisslert, M., Backert, S., Quiding-Jarbrink, M.
(2005). Helicobacter pylori Induces Transendothelial Migration of Activated Memory T Cells. Infect. Immun.
73: 761-769
[Abstract] [Full Text] -
Kranzer, K., Eckhardt, A., Aigner, M., Knoll, G., Deml, L., Speth, C., Lehn, N., Rehli, M., Schneider-Brachert, W.
(2004). Induction of Maturation and Cytokine Release of Human Dendritic Cells by Helicobacter pylori. Infect. Immun.
72: 4416-4423
[Abstract] [Full Text] -
Ishihara, S., Rumi, M. A. K., Kadowaki, Y., Ortega-Cava, C. F., Yuki, T., Yoshino, N., Miyaoka, Y., Kazumori, H., Ishimura, N., Amano, Y., Kinoshita, Y.
(2004). Essential Role of MD-2 in TLR4-Dependent Signaling during Helicobacter pylori-Associated Gastritis. J. Immunol.
173: 1406-1416
[Abstract] [Full Text] -
Wen, S., Felley, C. P., Bouzourene, H., Reimers, M., Michetti, P., Pan-Hammarstrom, Q.
(2004). Inflammatory Gene Profiles in Gastric Mucosa during Helicobacter pylori Infection in Humans. J. Immunol.
172: 2595-2606
[Abstract] [Full Text] -
Innocenti, M., Thoreson, A.-C., Ferrero, R. L., Stromberg, E., Bolin, I., Eriksson, L., Svennerholm, A.-M., Quiding-Jarbrink, M.
(2002). Helicobacter pylori-Induced Activation of Human Endothelial Cells. Infect. Immun.
70: 4581-4590
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»