Vol. 274, Issue 6, G992-G996, June 1998
H. pylori stimulates gastrin
release from canine antral cells in primary culture
Frank S.
Lehmann1,
Neal
Schiller2,
Timothy
Cover3,
Ritchard
Hatch2,
Rein
Seensalu1,
Kimitoshi
Kato1,
John H.
Walsh1, and
Andrew H.
Soll1
1 CURE: Gastroenteric Biology
Center, University of California, Los Angeles
90073; 2 University of
California, Riverside, California 92521; and3
Division of Infectious Diseases, Vanderbilt
University School of Medicine, Nashville, Tennessee 37232-2605
 |
ABSTRACT |
Patients chronically infected with
Helicobacter pylori are known to have
hypergastrinemia. Previous studies have demonstrated the stimulation of
gastrin from isolated G cells by monocytes and cytokines. The aim of
this study was to determine if H. pylori can directly stimulate gastrin secretion. The
secretion of gastrin from canine G cells in 48-h primary cultures was
investigated using either live H. pylori bacteria or various bacterial extracts from
three well-characterized strains. Whole bacterial sonic extracts and
water-extracted surface proteins, but not PBS extracts, from strains
43579 (CagA+/VacA+),
60190 (CagA+/VacA+),
and 60190:v1
(CagA+/VacA
)
significantly stimulated gastrin release. Controls demonstrated that
gastrin stimulation by the sonic extracts was not due to a direct toxic
effect on G cells. We conclude that H. pylori produces a soluble factor(s), which can directly
stimulate gastrin release in enriched canine G cell cultures. This
stimulatory effect may play an important role in the
H. pylori-associated hypergastrinemia and subsequent development of peptic ulcer disease.
Helicobacter; extracts; G cells
| |
INTRODUCTION |
HELICOBACTER PYLORI (HP) is the
causative agent of chronic superficial gastritis in humans, and
infection with this organism is a major factor contributing to the
pathogenesis of peptic ulcer disease (3). The mechanism by which HP
predisposes the duodenum to ulceration is currently unknown. One
attractive hypothesis is that the infection stimulates increased
release of gastrin, which in turn induces acid secretion, and that it
is this excessive duodenal acid load that causes ulceration (19).
Numerous studies have confirmed that duodenal ulcer patients as well as
asymptomatic subjects infected with HP have increased plasma gastrin
concentrations in the basal state (18) as well as after stimulation by
a meal (11), bombesin (12), or gastrin-releasing peptide (2). Eradication of the infection results in a marked fall in basal and
meal-stimulated gastrin release (11).
The mechanism(s) by which HP affects gastric endocrine cells is
unclear. It has been suggested that products from the bacterium itself,
inflammatory cells or cell products, or a combination of these might
influence endocrine cell function (10). Previously we reported the
stimulation of gastrin release from cultured canine G cells by
monocytes and tumor necrosis factor-
(TNF-) (15, 16). The
stimulatory effect of TNF- has now been confirmed in different G
cell preparations (1, 26). However, a direct effect of HP on gastrin
release has not been described. Using enriched canine G cell primary
cultures, we examined the effect of three well-characterized strains of
HP on gastrin release. G cells were incubated with either actively
motile exponential-phase cultures or bacterial extracts.
| |
METHODS |
Cell separation, enrichment, and culture.
Canine G cells were prepared and cultured from adult mongrel dogs as
previously described in detail (25). Briefly, the antral mucosa tissue
was separated from the submucosa and minced, and cells were dispersed
by sequential incubation with collagenase and EDTA. Dispersed cells
were washed, collected by centrifugation, and filtered through nylon
mesh. The cells were separated by velocity sedimentation using a
Beckman elutriator. Cell fractions that have been shown to contain
maximal gastrin immunoactivity (25) were collected, and elutriated
cells were centrifuged and resuspended in DMEM-F12, supplemented with
2% newborn calf serum, 100 µg/ml amikacin, 8 µg/ml insulin, 2 mM
glutamine, and 0.1 µg/ml hydrocortisone. Cells were plated on
Matrigel (Collaborative Research, Waltham, MA)-coated 24-well plates at
a concentration of 1 × 106
cells/well and incubated for 2 days in a humidified atmosphere of 5%
CO2-95% air at 37°C.
Gastrin release.
All release studies were performed using mucosal antral cells
maintained in short-term culture, which resulted in an enrichment of G
cells to 8-12% of the viable cell population (9). Somatostatin cells accounted for ~1.5% and mucous cells for the remainder. Before
release studies, each multiwell plate was washed twice with release
medium (9). Monoclonal somatostatin antibody CURE S6
(107 M) was included in all
experiments to rule out any indirect effect on gastrin stimulation from
somatostatin cells. S6 antagonizes the effects of exogenously
administered and endogenously released somatostatin in the stomach
(27). Somatostatin content in the G cell preparations was <5
pmol/106 cells (9), which is
blocked by S6 at a concentration of
107 M (27). After the
plates were incubated for 2 h at 37°C in 5%
CO2-95% air with either control
DMEM culture medium, live HP bacteria, or various bacterial extracts,
the G cell supernatants were collected and the cells were detached from
the base of unstimulated wells to determine the cell content of gastrin
(16). All release studies were performed in triplicate. Three wells on
each plate incubated without stimulant indicated basal release, and
three wells received 1011 M
bombesin as a positive control for gastrin release. The percentage of
gastrin release was then calculated by dividing the amount of gastrin
in the test supernatant by the amount remaining in the adherent cells
(9).
Gastrin assay.
Gastrinlike immunoactivity was measured by radioimmunoassay as
described previously, using antiserum 1611 in a final titer of 1:80,000
and tracer prepared by iodination of gastrin-17 by the chloramine-T
method. This assay recognizes all COOH-terminal fragments of gastrin
longer than four residues but does not detect glycine-extended gastrin
variants (25).
HP strains.
Three well-characterized strains were used for these studies:
1) American Type Culture Collection
(ATCC) strain 43579 (14), 2) strain
60190 (17), which is also known as ATCC 49503, and 3) strain 60190:v1, a
cytotoxin-deficient derivate of 60190 (7). Strains 43579 and 60190 were
initially isolated from human gastric biopsies and are positive for
CagA and vacuolating toxin, whereas 60190:v1 is an isogenic mutant in
which the VacA gene has been disrupted by insertion of a kanamycin
resistance gene (7).
Expression of CagA+ strain 43579 was assessed by
immunoblotting whole cells, using anti-CagA antiserum as described
previously (6). The vacuolating cytotoxic activity of strain 43579 was evaluated by incubating HP culture supernatants with HeLa cells for 18 h and monitoring cell vacuolation with the neutral red uptake assay, as
described previously (4). Exponential-phase broth cultures of each
strain were prepared by growth in Brucella broth (Difco, Detroit, MI)
containing 5% heat-inactivated FCS and 1% IsoVitaleX
(Becton-Dickinson Microbiology, Cockeysville, MD) at 37°C in a
humidified 12% CO2 incubator.
Previous growth curves, assessed by phase-contrast microscopy, optical
density, and bacterial colony determination, have demonstrated that
exponential phase growth occurs at about 36 h when cells are incubated
in the culture medium described above. The optical density of the cultures before use was determined to be 620 nm, and the organisms were
centrifuged and resuspended in DMEM at a concentration of 2 × 108 bacteria/ml. Fifty microliters
of this suspension were used per well of G cells, which gives a final
concentration of 107
bacteria/well. The cultures were actively motile as determined by
phase-contrast microscopy.
For preparation of bacterial extracts, these bacteria were cultured on
Brucella agar plates containing 5% sheep red blood cells and 1%
IsoVitaleX at 37°C in a humidified 12%
CO2 incubator. After growth for
either 48 or 72 h, the plates were swabbed into 0.15 M NaCl and the
optical density (620 nm) was determined and adjusted to 1.0 (5 × 108 bacteria/well). All of the
following bacterial extracts were prepared from this same number of
organisms, and the soluble extracts were stored at 
80°C
until use.
Sonic extracts were prepared by centrifuging the bacterial suspension,
resuspending the bacteria in PBS, and sonicating to complete disruption
(typically using 4 bursts of 140 W each for 30 s). Intact cells and
large bacterial fragments were removed by high-speed centrifugation at
45,000 g for 15 min at 4°C and 0.2-µm filtration. Water extracts were prepared following the protocol of Mai et al. (20) by centrifuging the bacterial suspension, resuspending the bacteria in sterile, double-distilled water, and
vortexing vigorously for 45 s, followed by centrifugation and
filtration. Finally, PBS extracts were prepared as described by Craig
et al. (8). After resuspension of the bacteria in PBS, the bacteria
were incubated at 37°C for 4 h, before centrifugation and
filtration.
A qualitative determination of urease activity was assayed by
incubating 100 µl of extract with 100 µl of urea test broth (24)
overnight in a 37°C water bath. The development of a pink-red color
indicated urease activity. All three of the preparations
sonic, water,
and PBS extractswere positive after overnight incubation. Protein
concentrations were determined using a modification of the Lowry
protein assay (21).
Responsiveness of G cells after release experiments.
The viability of G cells was assessed after release experiments with HP
sonicates to exclude a cytotoxic effect. The viability of G cells was
determined by restimulation with bombesin, as previously described
(16). Briefly, after completion of the incubation with sonic extracts,
the cells were washed and incubated for an additional 4 h at 37°C
in 5% CO2-95% air. G cells were
then stimulated with 1010 M
bombesin for 2 h. Previously unstimulated cells were also treated with
bombesin and used as a positive control.
Statistical analysis.
Statistical differences were assessed by Student's
t-test. Results are means ± SE.
P < 0.05 was considered significant.
| |
RESULTS |
HP live cells.
There was a modest but significant (P < 0.05, n = 5) stimulation of
gastrin release from canine G cells when cells were exposed to actively
motile, exponential-phase cultures of strain 43579 and 60190 at a
concentration of 107 bacteria/well
(the highest concentration tested) (Fig.
1). Strain 43579 stimulated gastrin release
by 19% above basal values, whereas strain 60190 caused a 28%
increase. Strain 60190:v1 also induced gastrin secretion, although the
results were not statistically significant. When tested at
106 and
105 bacteria/ml, no stimulatory
effect was induced by any of the strains.
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Fig. 1.
Effect of Helicobacter pylori (HP)
live bacteria on gastrin release. Basal gastrin release is compared
with gastrin release stimulated by HP live bacteria (strains 43579, 60190, and 60190:v1). Data are means ± SE from 5 preparations.
* P < 0.05. Basal gastrin
release was 0.7 ± 0.1%; bombesin-stimulated release was 2.6 ± 0.3% (not shown).
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HP sonic extracts.
Incubation of canine G cells with whole cell sonic extracts obtained
from 48-h plate-grown cultures of each strain significantly enhanced
gastrin release (P < 0.01, n = 5). There was a modest but still
significant increase induced by 72-h broth cultures from strain 43579 but not the other two strains (Fig. 2).
Strain 43579 stimulated gastrin release by 53% (48 h) and 37% (72 h) above basal values, strain 60190 stimulated gastrin release by 32% (48 h) and 18% (72 h) above basal values, and strain 60190:v1 stimulated
gastrin release by 50% (48 h) and 3% (72 h) above basal values. The
protein concentration of all sonic extracts was 0.3 ± 0.1 mg/ml (48 h) and 1 ± 0.2 mg/ml (72 h).
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Fig. 2.
Effect of HP sonic extracts on gastrin release. Basal gastrin release
is compared with HP sonicate-stimulated release. Data are means ± SE from 5 preparations. * P < 0.05, ** P < 0.01. Basal
gastrin release was 0.8 ± 0.1%; bombesin-stimulated release was
2.6 ± 0.4% (not shown).
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Water-extracted surface components.
Extracts from each strain prepared by vigorous vortexing in
double-distilled water were able to cause significant
(P < 0.01, n = 5) stimulation of gastrin release,
regardless of whether the bacteria were cultured for 48 or 72 h (Fig.
3). This was all the more impressive since
the preparations contained less than 10 µg of protein/ml. Strain
43579 stimulated gastrin release by 41% (48 h) and 25% (72 h), strain
60190 by 41% (48 h) and 53% (72 h), and strain 60190:v1 by
33% (48 h) and 49% (72 h) above basal values. The bacteria
remained viable after water extraction as determined by phase-contrast
microscopy and colony-forming unit determination.
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Fig. 3.
Effect of HP water-extracted surface proteins on gastrin release. Basal
gastrin release is compared with release stimulated by water extracts.
Data are means ± SE from 5 preparations.
* P < 0.02, ** P < 0.01. Basal gastrin
release was 0.7 ± 0.1%; bombesin-stimulated release was 2.2 ± 0.3% (not shown).
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PBS extracts.
There was no effect on gastrin release when PBS extracts prepared from
48- or 72-h cultures from any of the three strains were added to the
monolayers (0.7 ± 0.1% basal vs. 0.7 ± 0.1 with PBS extracts,
n = 5). The protein concentration of
the PBS extracts was <10 µg/ml (48 h and 72 h).
Responsiveness of G cells after release experiments.
As a control for confirming cell viability after treatment with sonic
extracts, the responsiveness of the treated G cells to bombesin
stimulation was determined. Previously unstimulated G cells showed a
1.5-fold increase of gastrin release after bombesin stimulation,
similar to cells that had been previously exposed to HP sonic extracts
(n = 3; Fig.
4).
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Fig. 4.
Responsiveness of G cells after release experiments. G cells that had
been exposed to HP sonic extracts, as well as previously unstimulated
cells, were stimulated with
1010 M bombesin. Open bars,
basal values; solid bars, bombesin-stimulated levels. Data are means
from 3 preparations.
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| |
DISCUSSION |
We report here for the first time that live HP bacteria or bacterial
extracts can directly stimulate the release of gastrin from canine
antral G cells. These experiments demonstrate that HP produces a
soluble factor(s), probably surface exposed, which can cause G cell
cultures to release enhanced levels of gastrin. This observation is
consistent with the hypothesis that HP can directly contribute to the
damage observed in gastroduodenal disease.
It is now well established that duodenal ulcer patients chronically
infected with HP have increased basal and stimulated gastrin release.
Circulating gastrin may be responsible for driving basal acid
secretion, which is increased in duodenal ulcer patients (19).
Recently, it was demonstrated that the fall in gastrin after HP
eradication was accompanied by a proportional fall in basal acid
secretion (22). Reversion of hypergastrinemia and increased basal
secretion may contribute to the duodenal ulcer remission after HP
eradication.
The presence of HP in the gastric antrum is associated with
mucosal inflammatory cells such as neutrophils, lymphocytes,
monocytes/macrophages, and plasma cells (3, 20). HP secretes a
potent chemotactic factor, possibly urease, for mononuclear and
polymorphonuclear inflammatory cells and stimulates monocytes to
release several cytokines, including TNF- (8, 13). In a previous
study we demonstrated gastrin secretion in isolated G cells by
stimulated monocytes and TNF- (16). The inflammatory response
induced by HP may in turn stimulate monocytes and TNF- secretion,
which may have an additional impact on G cell function.
Water-extracted surface proteins stimulated gastrin release in our
experiments. Mai et al. (20) found that this extraction procedure had
essentially no contamination by lipopolysaccharide. Because these
proteins are shed from the bacterial cell, this material may be similar
to that released from HP in vivo (20). HP usually does not appear to
invade the mucosa, and therefore the release of soluble proteins could
be an important factor in stimulating gastrin release. Our observation
that actively motile intact HP stimulate gastrin release is consistent
with this hypothesis.
We examined bacterial extracts at two different time points to
determine whether the bacterial gastrin stimulatory factor(s) was
produced primarily in the midexponential (48 h) or early stationary (72 h) phase. Although the water extracts were equally potent in gastrin
stimulation using either 48- or 72-h cultures, the sonic extracts
prepared at 48 h were more effective than those prepared at 72 h.
Although the explanation for this remains unknown, one possibility is
that cytoplasmic or periplasmic proteases produced during later growth
stages may be released by sonication and may alter the gastrin
stimulatory activity. Additional studies are planned that will
characterize the gastrin-stimulating factor(s) present in
water-extracted surface proteins from these HP strains.
Two major phenotypic characteristics known to differ among HP strains
are production of a vacuolating cytotoxin (5) and the presence of a
cytotoxin-associated protein encoded by CagA (6). These two related
phenotypes are considered to be potentially important virulence factors
that may affect the clinical outcome of HP infection (7). The
mechanisms whereby CagA or vacuolating toxin could be related to the
pathogenesis of ulcer disease are unknown. Restimulation of G cells
with bombesin after treatment with sonic extracts demonstrated that
these monolayers were still viable and intact, indicating that
stimulation of gastrin release occurred without cell damage by enzymes
or cytotoxic activity. In addition, disruption of the VacA gene did not
influence the stimulatory effects of sonicates and water extracts on
gastrin release in our experiments, suggesting that the importance of this cytotoxin as a virulence factor is unrelated to its potential to
stimulate gastrin release.
Our whole organism studies provided only modest increases of gastrin
stimulation over baseline at the highest level of bacteria we used
(107/ml) for the two
VacA+ strains, whereas at lower
concentrations no stimulatory effect was induced by any of the strains.
HP live cells of strain 60190:v1 also induced gastrin release, although
the results were not statistically significant. It is probable that at
higher concentrations of the VacA-deficient strain, we would have seen
significant levels of gastrin secretion. A dose-response curve with
higher concentrations was not performed, because increasing bacterial
numbers to 108/ml or more might be
much greater than physiologically reasonable.
Eradication of HP in infected patients results in increased synthesis
and release of somatostatin (23). It has been suggested that
suppression of somatostatin might explain the increased gastrin release
in HP-infected patients (23). However, the stimulatory effect on
gastrin release by HP observed in this study occurred in the presence
of the somatostatin antibody S6, suggesting that HP causes a direct
effect on G cell-mediated gastrin release. The stimulatory effect on
gastrin release by mononuclear cells and cytokines has also been
observed with S6 (1, 16). However, these observations do not exclude an
additional, independent effect on somatostatin release.
We have provided evidence that three HP strains, incubated with canine
G cell primary cultures as either actively motile bacteria, soluble
sonic cell extracts, or water-extracted surface proteins, stimulate
gastrin release. HP and cytokines may both play an important role in
HP-induced hypergastrinemia.
| |
FOOTNOTES |
Address for reprint requests: F. Lehmann, Division of
Gastroenterology, Univ. Hospital of Basel, 4031 Basel, Switzerland.
Received 25 April 1997; accepted in final form 10 February 1998.
| |
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Abstract
Introduction
