1 Department of Medicine II and 3 Krankenhaus München Bogenhausen, Technical University of Munich, Munich; and 2 Anatomisches Institut der Universität München, München, Germany
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ABSTRACT |
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Gastric
Helicobacter pylori infection may lead to multifocal
atrophic corpus gastritis associated with loss of epithelial cells as
well as glandular structures. The current work investigated H. pylori effects on cell death of isolated, nontransformed rat parietal cells (PC). Highly enriched rat PC (>97%) were isolated from
gastric mucosa and cultured in serum-free medium over 24 h. The
cells were cocultured over 8 h with cytotoxin-associated immunodominant protein (cagA)+/vacuolating toxin
(vacA)+ or with cagA/vacA
H. pylori laboratory strains and also with H. pylori mutants deleted in several genes of the cag pathogenicity
island. Staphylococcus aureus or Campylobacter
jejuni were used as controls. Apoptosis was
determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and electron microscopy. Interleukin (IL)-8 and
cytokine-induced neutrophil chemoattractant (CINC)-1 secretion was
measured by ELISA. Activation of nuclear factor-
B (NF-
B) was
studied in nuclear extracts of PC by electrophoretic mobility shift
assay. Apoptosis of PC was induced in a concentration- and time-dependent manner by cagA+/vacA+ H. pylori strains but not by cagA
/vacA
negative strains or by the cagE knockout mutant. S. aureus
and C. jejuni had no effect. PC showed no IL-8 or CINC-1
secretion on exposure to cagA+/vacA+ H. pylori. cagA+/vacA+ strains induced
activation of NF-
B complexes in nuclear extracts of PC, which were
composed of p65 and p50 subunits. No significant stimulation of
NF-
B activation was detected by incubation of PC with the cagE
knockout mutant. Preincubation of PC with antisense but not missense
oligodeoxynucleotides against the p65 subunit significantly reduced DNA
binding to the
B recognition sequence. The p65 oligonucleotides as
well as the proteasome inhibitor
N-CBZ-isoleucin-glutamin-(o-t-butyl-)-alanin-leucin and the nitric oxide synthase inhibitor
NG-monomethyl-L-arginine completely
prevented PC apoptosis induced by
cagA+/vacA+ strains. In summary, cagE presence
appears to be essential for H. pylori-induced
apoptosis of gastric parietal cells, and this effect is
dependent on the activation of NF-
B and production of nitric oxide.
nuclear factor-B
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INTRODUCTION |
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THE GASTRIC MUCOSA IS FREQUENTLY infected with the gram-negative bacterium Helicobacter pylori, colonizing the antral part of the gastric epithelium in which the pH is elevated to 3-4, which allows optimal growth (7). Previous studies have revealed that urease activity of H. pylori depends on urea uptake through a specific channel, which is open at pH values between 3 and 6 but closes at levels >7 (6, 19, 28). During long-term infection as well as long-term therapy with proton pump inhibitors, the infection may also proceed to the gastric corpus, resulting in atrophy of gastric glands, hypochlorhydria, and possibly the development of gastric adenocarcinoma (14, 32, 33).
One mechanism by which H. pylori generates mucosal atrophy
may be the induction of apoptosis of the epithelial mucosal
barrier followed by destruction of the remaining mucosa by exposure to luminal acid. Indeed, in vivo and in vitro studies demonstrate that
infection with H. pylori is associated with
apoptosis of gastric epithelial cells (20, 31);
coculture of human gastric cancer cell line (AGS) cells with H. pylori in vitro resulted in growth inhibition predominantly at the
G(0)-G(1) checkpoint, possibly mediated
by changes of the regulatory proteins p53, p21, and cyclin E
(1). Another mechanism may be a more indirect effect. These studies have suggested that adherence of H. pylori to the gastric mucosa triggers the production of
proinflammatory cytokines such as interleukin (IL)-8, tumor necrosis
factor (TNF)-, and IL-1
(12). The release of these
cytokines in turn induces the accumulation of inflammatory cells and
also leads to a sustained functional impairment of mucosal cells. For
example, incubation of parietal cells (PC) and enterochromaffin-like
cells with IL-1
induced apoptosis of these cells that are
crucial for acid secretion (9, 12, 18, 25, 26).
It may also be of special interest to investigate direct effects of the bacteria on gastric PC. As mentioned above, long-term infection as well as long-term therapy with proton pump inhibitors may lead to the colonization of the gastric corpus, and the only spaces in which the bacterium may survive appear to be the acidic spaces inside of the gastric gland, where the bacteria are in close contact with the acidic lumen of parietal cells. Indeed, pathologists have detected bacteria inside the gastric gland; it may therefore be possible that the bacteria affect PC directly by interfering with the life cycle of this important cell type.
Some H. pylori strains appear to be more virulent than others and may, therefore, also affect PC differentially. Several virulence factors of H. pylori, especially the cag pathogenicity island (cagPAI), encode type IV secretion machinery. Products encoded by the cagPAI, such as CagA or CagE, have been associated with increased mucosal inflammation (9, 12, 34). Also, H. pylori strains expressing vacuolating toxin (VacA) have been considered to be associated with increased pathogenic potential of the bacterium (4, 5).
Our current work investigated direct effects of H. pylori on
programmed cell death in gastric PC. Our results show a significant stimulation of apoptosis, which appears to depend on the
presence of the cagE (also called picB) gene (34). It
appears that the effect of H. pylori involves the generation
of nuclear factor-B (NF-
B) but not the generation of the rat IL-8
homolog cytokine-induced neutrophil chemoattractant (CINC)-1.
In this sense, H. pylori has a direct effect on
parietal cells that is distinct in its signal transduction in
epithelial cells, in which IL-8 is released in large amounts. Our data
thus contribute to the understanding of gastric atrophy and
carcinogenesis and may present a model for the events during late
stages of mucosal destruction.
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MATERIALS AND METHODS |
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Cell isolation and primary cell culture. Highly enriched rat gastric PC were prepared as previously described (27). A total of 80 preparations was used (5 rats/preparation). All killing experiments were performed in accordance with the ethical guidelines of the Technical University of Munich. The experiments comply with all relevant local and institutional regulations. Briefly, the stomachs were treated with pronase E (1.3 mg/ml; Roche, Mannheim, Germany) for enzymatic digestion, and the dispersed cells were then subjected to counterflow elutriation (JE-6 elutriation rotor, Beckman Instruments, Palo Alto, CA) and density gradient centrifugation. Enriched PC were placed on six-well plates precoated with Matrigel diluted 1:500 with sterile dH2O (Becton Dickinson, Heidelberg, Germany). For immunocytochemistry and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, 4-5 × 106 cells were grown on sterile glass coverslips coated with Cell-Tak (Becton Dickinson; dilution 1:1 with 0.5 M NaHCO3). Cells were cultured in DMEM (GIBCO BRL, Eggenstein, Germany) supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, and 5 µg/ml sodium selenite, 100 µg/ml gentamicin, 10 µg/ml hydrocortisone, and 1 g/500 ml BSA. For stimulation experiments, PC were incubated in serum-free medium. Cell purity in this cell culture system was >97%, as measured by Giemsa staining. Cell viability was determined by trypan blue staining.
Bacterial strains and infection.
H. pylori isolates used in this work were as follows: G27
and HP2808, both wild-type laboratory strains and both expressing the
cagPAI and the vacA genes, and G27 knockout
mutants were used. These strains were deficient for the
cagE, cagF, cagH, cagN, or the cagA gene. The wild-type strain and the isogenic mutants
were kindly provided by A. Covacci, Immunobiological Research
Institute, Chiron, Siena, Italy. Tx30 is a laboratory strain lacking
the cagPAI and vacA. The isolates were kept at 80°C in Brucella
broth with 10% fetal bovine serum containing 10% (vol/vol) glycerol. H. pylori strains were cultured on WC-agar plates containing
10% horse serum in a microaerophilic atmosphere at 37°C over
48-72 h. Bacteria were harvested in PBS (pH 7.4) and diluted
corresponding to the multiplicity of infection (MOI) wanted, using a
McFarland densitometer. The scale is graduated in both McFarland
standard units and the average bacterial concentrations. The viability of bacteria was monitored by light microscopy. The parietal cells cultured in DMEM at 37°C were then incubated with the bacteria. In
some experiments, H. pylori strain G27 was heat inactivated (30 min, at 60°C), sonicated, or treated with chloramphenicol (25 µg/ml for 30 min). S. aureus and C. jejuni were
used as controls. They were grown in their corresponding environment
and were as equally diluted as the H. pylori strains.
Electrophoretic mobility shift assay.
For electrophoretic mobility shift assay (EMSA), nuclear extracts (NE)
of enriched PC were used. Per sample, 1 × 107 cells
were incubated with H. pylori over 8 h. Nuclear
extracts were prepared as follows. Briefly, parietal cells were
detached from the tissue culture plate with 1 × trypsin-EDTA and
washed once in ice-cold PBS. After centrifugation (1,000 rpm, 5 min), they were resuspended in buffer 1 (15 mM NaCl, 15 mM
-mercaptoethanol, 15 mM Tris · HCl, 2% protein inhibitor
cocktail, and 0,5% phenylmethylsulfonyl fluoride, all from Sigma,
Munich, Germany). After centrifugation, cells were resuspended in
buffer 1 and kept on ice for the next 30 min. They were
centrifuged again and resuspended in buffer 2 (buffer
1 supplemented with 2 mM EDTA, 0.5 mM EGTA, and 0.34 M sucrose).
The lysates were transferred into special tubes and homogenized over 11 min to obtain the nuclei. After saccharose density gradient
centrifugation with buffer 3 (buffer 1 supplemented with 1 mM EDTA, 0.25 mM EGTA, and 1.37 M sucrose), the
pellets were resuspended in binding buffer (250 mM HEPES, 5 mM EDTA, 20 mM 1,4-dithiothreitol, 40% glycerol, 1 M NaCl) and sonicated (Branson Sonifier, Heidelberg, Germany). NE were immediately placed on dry ice
and stored at
80°C. For EMSA, 10 µg nuclear protein in 20 µl
H2O and binding buffer were incubated with 4 µg of
poly-(dI:dC) for 5 min at room temperature. Then, 25 ng of nonlabeled
(cold) oligonucleotides (ODN) containing the HIV-NF-
B consensus
sequence or the SP-1 sequence (Gelshift assay kit; Stratagene,
Cambridge, UK) were incubated over 20 min with the lysates of
unstimulated cells for competition assay. Finally, 2 µl of
32P-labeled oligos (~100,000 counts/min, labeled
with T4 polynucleotide kinase, Roche) containing the NF-
B consensus
site or the SP-1 site were added to each probe for another 20 min. The
probes were kept on ice, mixed with 2.2 µl of sample buffer [20%
Ficoll 400 and 0.25% bromphenol blue in Tris-borate EDTA (TBE)] and
loaded on a 7% acrylamide gel. Electrophoresis was carried out at room temperature for 7-10 h with TBE. After completion of the
electrophoresis, the gel was washed in H2O, dried, and
exposed to either an X-ray film at
70°C for 2-5 days or to a
phosphor imaging screen (Molecular Dynamics; Sunnyvale, CA) for ~2 days.
Transfection with antisense-phosphorothioate ODNs.
Antisense (AS) ODNs spanning the translation initiation codon were
constructed for the NF-B subunit p65 (5'-ggg gaa cag ttc gtc cat
ggc-3'). Missense (MS) ODN (5'-ggg gcg atg agg cct act atc-3') did have
an identical nucleotide content in random order and served as negative
control. ODN were synthesized in a phosphorothioate-modified form by
MWG Biotech (Munich, Germany). For use in internalization studies, ODN
were 5'-labeled with 6-carboxyfluorescein (6-FAM). Freshly prepared
parietal cells were grown on Cell-Tak coated slides at a density of
4-5 × 105 cells/slide in culture media over
8 h to allow recovery. Cells were incubated at 37°C over 8 h with 10 µM FAM-labeled, AS or MS ODN. Transfection efficacy was
examined with FAM-labeled ODN by fluorescence microscopy. More than
85% of the cells showed green fluorescent staining in the nuclear region.
Determination of parietal cell apoptosis.
To determine PC apoptosis, a TUNEL reaction was used. For this
experiment, PC were first grown on glass slides for 12 h as described and then bacteria were added at the indicated MOI. Incubation was performed at the indicated time periods. The stimulation was ended
by fixation in Bouin's solution over 15 min. Cells were lysed and
processed to block unspecific binding sites and destroy endogenous
peroxidase activity. Incubation with biotinylated deoxynucleotides allowed terminal transferase-mediated labeling of PC-DNA strand breaks
caused by apoptosis. A peroxidase reaction (Vector
Laboratories, Burlingame, CA) was used to stain the labeled cell nuclei
for light microscopy. Cells were counted in visual fields containing more than 100 cells; the percentage of apoptotic cells was obtained by dividing the number of cells with positive staining by the total
number of cells. Apoptotic rates were determined under basal conditions, addition of different bacteria, or under the simultaneous addition of various inhibitors. In some experiments of Fig. 8, a
proteasome inhibitor was used preventing degradation of the NF-B
complex. N-CBZ-isoleucin-glutamin-(o-t-butyl-)-alanin-leucin (PSI) is a tetrapeptide N-Ile-Glu-Ala-Leu (obtained from
Sigma). The nonspecific nitric oxide (NO) synthase (NOS) inhibitor
NG-monomethyl-L-arginine
(L-NMMA) was purchased from Sigma.
Electron microscopy. Cells were fixed with 4% paraformaldehyde/0,5% glutaraldehyde and postfixed with 4% OsO4/potassium hexacyanoferrate. After being embedded in Epon, thin sections were cut, contrasted with uranylacetate (2%)/lead citrate (2.7%), and examined with an EM10 electron microscope (Carl Zeiss, Jena, Germany) as described previously (8).
Statistical analysis. Results are shown as the means ± SE. Data were analyzed by one-way ANOVA followed by Newman-Keuls test. P values <0.05 were considered significant.
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RESULTS |
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Induction of apoptosis in highly enriched PC by incubation
with H. pylori.
After 12 h of culture, purified rat gastric PC were incubated with
the H. pylori strain G27 over 8 h in regular media.
Bacterial densities were examined by McFarland assay. At the indicated
MOI of 2, a concentration of two bacteria per cell was used. As
visualized in the Fig. 1A, a
significant stimulation of apoptosis was already observed at an
MOI of 10 corresponding to 10 bacteria per cell. At higher
concentrations, the stimulation was even more prominent (P < 0.01). Maximal effects were observed at an MOI of
20 (n = 5 independent experiments).
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Specificity of H. pylori effects.
To demonstrate the specificity of the H. pylori effects,
several other bacteria were added, and their effect on PC was
investigated. As depicted in Fig.
2A, TNF- and the H. pylori strain G27 induced a twofold increase of apoptosis
of PCs after 8 h of incubation. In contrast, S. aureus
and C. jejuni did not significantly induce programmed cell
death at an MOI of 10 (n = 4 experiments). Next, different H. pylori strains characterized by the presence or
absence of the vacA and cagA gene were incubated
simultaneously with enriched PC. As shown in Fig. 2B,
TNF-
at 20 ng/ml, the laboratory strains H. pylori G27 as
well as the H. pylori laboratory strain 2808 at an MOI of 10 induced apoptosis two- to threefold compared with the basal
rate. In contrast, the H. pylori strain Tx30 lacking the
vacAs1 and cagA genes did not exert any
significant effect on PC apoptosis. To determine the effect of
heat inactivation, the H. pylori G27 strain was sonicated or
heat inactivated over 30 min. The procedures were used to discriminate
between effects of lipopolysaccharides (LPS) present in the bacteria or
potential proteins. As shown in Fig. 2C, sonication and heat
inactivation at 60° over 30 min completely abolished the effects,
indicating that LPS are not mediating these effects. Similarly,
addition of chloramphenicol at 25 µg/ml over 30 min completely
prevented the effect of G27 on PC apoptosis.
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Costimulatory effects of H. pylori and TNF-.
Highly enriched PC were cultured and incubated with the H. pylori strain G27 over 8 h in the presence or absence of
TNF-
at 20 ng/ml. As indicated in Fig.
3A, both TNF-
and the
H. pylori strain G27 alone had stimulatory effects, and the
simultaneous addition produced an additive effect. As depicted in Fig.
3B, the G27 effect on PC apoptosis was not inhibited
by the simultaneous addition of the TNF-receptor antibody, whereas this
antibody abolished the effect of TNF-
. Thus the G27 effect was
not mediated by the release or action of TNF-
.
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Effects of knockout mutants deficient in genes of the cagPAI.
Different mutants of the cagPAI were used to examine the importance of
the proteins encoded by the genes. These mutants were provided by A. Covacci (Siena, Italy). As shown in Fig.
4, the cagA, cagH, cagF, and cagN mutants
stimulated apoptosis of PC after 8 h of incubation. In
contrast, addition of the cagE mutant did not induce apoptosis
of rat gastric PC, indicating that this effect depends on the cagE
protein.
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Signal transduction in H. pylori-induced apoptosis of PC.
The G27 strain was used to stimulate apoptosis in PC after
8 h of coculture. p65 AS ODN (100 µM) were used to block the
effect of NF-B. As shown in Fig. 5,
the AS ODN completely inhibited the effect, whereas the MS ODN did not
block G27-induced programmed cell death. In the experiments shown in
Fig. 5, the transfection rates were 84 (±6) % in three different
experiments. The apoptotic rate was 15% (±3), and the rate in the
H. pylori incubated fraction was 35% (±2), indicating that
the majority of cells that die was also transfected with ODN.
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Gel shift assays of H. pylori-induced activation of NF-B in PC.
PCs were incubated with vehicle, the H. pylori strain G27,
or the G27 knockout mutants cagH, cagA, cagE, cagF, and cagN. Nuclear extracts were prepared, and EMSA was performed as described above. The
extracts were incubated with radiolabeled, double-stranded ODNs
containing the NF-
B consensus sequence. A total of three independent experiments was performed using different strains mutated
in the cagPAI. As indicated in Fig.
6A, the G27 strain strongly
activated the binding of NF-
B to the consensus sequence (Fig.
6A, lane 2). Only the cagE mutant failed to
induce NF-
B binding to the consensus sequence (lane 5).
Densitometric analysis of three independent experiments was used to
quantitate the intensity of both bands relative to the intensity under
basal conditions (Fig. 6B), indicating a strong stimulation
with most strains but a lack of stimulation using the cagE mutant
(lane 5, Fig. 6B). As the control experiment,
EMSA was also performed with Sp-1 binding to the corresponding
consensus sequence, but no changes were observed; densitometric
analysis did not yield significant differences (Fig. 6, C
and D). As a further control, a supershift assay was
performed with antibodies against the p50 and p65 subunits of NF-
B
to confirm the specificity of the EMSA; both antibodies shifted the
bands of the two NF-
B subunits to higher molecular weights (Fig.
6E).
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Effect of AS ODNs on NF-B activation.
PC cells were preincubated overnight with AS ODNs for the NF-
B
subunit p65 and its MS ODN form serving as negative control (Fig.
7, A and B). After
culture medium change, the cells were again incubated with strain G27,
which activated the binding of NF-
B to its consensus sequence. The
addition of the AS, but not the MS ODN prevented this effect.
Densitometric analysis (Fig. 7B) inhibited binding of the
NF-
B AS ODN in three independent experiments, whereas intensities
for the Sp-1 bands were not significantly affected by addition of the
AS ODNs (Fig. 7, C and D).
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Effects of proteasome inhibitors and L-NMMA on
apoptosis.
Rat gastric PCs were incubated with vehicle or the H. pylori
strain G27 over 8 h, and apoptosis was measured using
TUNEL assay. Simultaneously, the proteasome inihibitor PSI (1 mM) or
the NOS inhibitor L-NMMA (104 M) was added.
As indicated in Fig. 8, both inhibitors
prevented the induction of apoptosis in rat PCs induced by the
addition of the H. pylori strain G27.
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DISCUSSION |
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H. pylori colonizes the gastric corpus mucosa, and this
bacterial expansion often results in multifocal atrophic gastritis. During this long-term infection, H. pylori may also colonize
the crypts of the gastric glands, and a direct contact of the bacterium may be given, enabling direct interaction of H. pylori also
with PCs. In the current study, we were able to show a direct effect of
the bacteria on gastric PC. Culturing bacteria together with the
acid-producing cells resulted in programmed cell death at MOI rates of
1-20, as shown by TUNEL and specific features determined by EM.
This effect can be considered specific, because it was observed with
defined bacterial subtypes, it was not affected by antibodies against
cytokines known to affect PC function (such as TNF-), and the effect
was abolished by short-term heat inactivation, indicating that LPS or
related structures do not contribute to the H. pylori-induced apoptosis of PC. Addition of
chloramphenicol completely prevented the effects of live H. pylori on apoptosis, indicating that de novo bacterial
protein synthesis, for example, active production of proteins
potentially involved in this effect, is required during this process.
Incubation with H. pylori-induced apoptosis in a
terminally differentiated cell. PCs originate from stem cells, and
their approximate lifespan is <1 wk. Incubation may effectively alter the equilibrium between differentiation and apoptosis at a
specific cellular signal-transduction step. Previous studies have
revealed that H. pylori leads to a G1 arrest of a gastric
epithelial cell line (1). H. pylori initiates
epithelial cell signaling events that resulted in activation of the
transcription factor NF-B (21). Direct inhibition of
NF-
B abolished H. pylori-stimulated apoptosis in
epithelial cells; H. pylori activated NF-
B, which was
blocked by cotreatment with peroxisome proliferator-activated receptor
(PPAR)-
agonist (16). These results suggested that activation of a PPAR-
pathway attenuates the ability of H. pylori to induce NF-
B-mediated apoptosis in gastric
epithelial cells. In the current study, we also observed
apoptosis in PCs dependent on the activation of NF-
B.
Specific AS ODNs and a proteasome inhibitor PSI completely prevented
the H. pylori effects, indicating that NF-
B has a
proapoptotic function similar to observations made in other cell
types (10, 15).
In human epithelial cells, activated NF-B translocates to the
nucleus, where it upregulates IL-8 gene transcription
(30). Previous studies have shown that H. pylori infection activates IL-8 gene expression in gastric
epithelial cells in vitro and in vivo (2). IL-8 mRNA and
protein levels are increased in the gastric mucosa of patients with
H. pylori gastritis, and immunohistochemical studies
demonstrate increased IL-8 protein in gastric epithelial cells from
infected individuals (3, 12). H. pylori also
increases IL-8 mRNA levels and protein production in cultured
monolayers of AGS cells and other gastric epithelial cell
lines (2). Gene expression of IL-8, in turn, was
considered to be of crucial importance for the attraction of
granulocytes and monocytes, leading to the release of products that
affect epithelial cell death.
In the current study, we observed that H. pylori did not induce IL-8 production in rat gastric PCs. This may not be surprising, because in rats, no gene sequence has been detected thus far encoding for IL-8. Thus this finding may only represent a species difference between humans and rats; however, we also measured concentrations of the rat IL-8 homolog CINC-1 (36) in the supernatants of rat gastric PCs in the presence and absence of H. pylori. Because we did not detect CINC-1 in rat PCs but in rat mononuclear blood cells (positive control), it may be concluded that the signaling cascade activated by H. pylori does not involve a signal pathway associated with the transcription of the rat IL-8 homolog CINC-1.
It appears that the differential activation process for NF-B and the
rat IL-8 homolog CINC-1 in PCs may reflect different cellular
transduction mechanisms. Indeed, recent studies reported that H. pylori, when cocultured with AGS epithelial cells, rapidly activated MAP kinases (17). With the use of
specific inhibitors, the authors found that p38 and MEK-1 activity was
required for H. pylori-induced IL-8 production, but
activation of these proteins was not essential for H. pylori-induced NF-
B activation in epithelial cells
(17). Thus it may be possible that the missing effect on
IL-8/CINC-1 secretion in PCs may be the missing activation of p38 and
MEK-1 in PCs too, and activation of NF-
B may require other kinases
involved in the induction of cell death.
Our data further support the view that the cagE (picB) protein encoded
by the cagPAI is responsible for the induction of this programmed cell
death. The cagPAI of H. pylori is a 40-kb region immediately
upstream from the cagA gene that encodes >40 putative bacterial proteins. On the basis of sequence homology, the cagPAI encodes a secretion system that is involved in the export or surface expression of bacterial virulence factors (11). Gene
products of the cagPAI are also known to participate in epithelial cell activation by H. pylori. H. pylori
cagA+ strains are more potent in activating epithelial cell
IL-8 production than cagA bacteria (9, 23, 24,
24). H. pylori picB, a homolog of the Bordetella
pertussis toxin secretion protein, is required for induction of IL-8 in
gastric epithelial cells (34). Furthermore, disruption of
specific cag region genes markedly reduces H. pylori-mediated tyrosine phosphorylation of gastric epithelial
cell proteins, NF-
B activation, and IL-8 gene transcription
(21, 22, 29).
H. pylori strains deficient in cagE, but not mutated in the
cagA sequence, did not induce apoptosis of PC in the current
work. Similar observations regarding a differential effect of
cagE-deficient mutants on cellular signal transduction were also
observed by Keates at al. (17). The authors reported
differential activation of mitogen-activated kinases and c-jun kinases
by cagA+- and cagE-deficient H. pylori, similar
mechanisms may also be present in gastric PC. Thus c-Jun kinases may
also play an important role in the activation of NF-B in rat gastric
PC too.
A specific inhibitor of a possible NF-B downstream target was
further investigated. As indicated, the NOS inhibitor
L-NMMA completely inhibited the effect of H. pylori on apoptosis determined by TUNEL reaction in PCs.
These results indicate that a possible downstream target of NF-
B is
gene expression of NOS and NO production, thereby leading to DNA
breakdown. Similar observations were made in pancreatic
-cells
following IL-1
stimulation. In
-cells, NF-
B activation is
linked to apoptosis, and this effect appeared to be mediated by
induction of inducible NOS (iNOS) and generation of NO
(13). The promoter region of the rat iNOS gene contains the NF-
B consensus sequence at several positions (bp 71-79,
134-142, 888-896, and 930-938), underlining the
functional interaction between NF-
B activation and iNOS
transcription. Our data therefore support the idea that iNOS may be a
key enzyme mediating H. pylori-induced cell death, leading
to the generation of NO and subsequent apoptosis in PCs.
In summary, our data suggest that gastric PCs may be directly affected
by H. pylori, that this effect is mediated by the cagE product, and that it involves activation of the transcription factor
NF-B and the generation of NO.
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ACKNOWLEDGEMENTS |
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The wild-type strain and the isogenic mutants were kindly provided by A. Covacci, Immunobiological Research Institute, Chiron, Siena, Italy.
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FOOTNOTES |
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This work was supported by the Else Kröner Fresenius Stiftung (to B. Neu), Kuratorium für Klinische Forschung der TU München (KKF-8733156 to C. Prinz), Deutsche Forschungsgemeinschaft (DFG-411/7-1 to C. Prinz), and Gastrofoundation, Munich, Germany.
C. Prinz is a recipient of the Heisenberg Award from the Deutsche Forschungsgemeinschaft.
Address for reprint requests and other correspondence: C. Prinz, II. Medizinische Klinik, Technische Universität München, Ismaningerstr. 22, 81675 München (E-mail: christian.prinz{at}lrz.tum.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00546.2001
Received 27 December 2001; accepted in final form 19 March 2002.
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