1 CURE: Gastroenteric Biology
Center, 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
Helicobacter; extracts; G cells
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- 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
(10 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).
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
)
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.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
(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
Top
Abstract
Introduction
Methods
Results
Discussion
References
7 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
10
7 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 10
11 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).
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.
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RESULTS |
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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.
|
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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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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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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DISCUSSION |
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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.
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FOOTNOTES |
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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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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Beales, I. L.,
L. Post,
J. Calam,
T. Yamada,
and
J. Del Valle.
Tumour necrosis factor- stimulates gastrin release from canine and human antral G cells: possible mechanism of the Helicobacter pylori-gastrin link.
Eur. J. Clin. Invest.
26:
609-611,
1996[Medline].
2.
Beardshall, K.,
S. Moss,
J. Gill,
S. Levi,
P. Ghosh,
R. Playford,
and
J. Calam.
Suppression of Helicobacter pylori reduces gastrin releasing peptide-stimulated gastrin release in duodenal ulcer patients.
Gut
33:
601-603,
1992[Abstract].
3.
Blaser, M. J.
Hypotheses on the pathogenesis and natural history of Helicobacter pylori-induced inflammation.
Gastroenterology
102:
720-727,
1992[Medline].
4.
Cover, T. L.,
P. Cao,
C. D. Lind,
K. T. Tham,
and
M. J. Blaser.
Correlation between vacuolating cytotoxin production by Helicobacter pylori isolates in vitro and in vivo.
Infect. Immun.
61:
5008-5012,
1993[Abstract].
5.
Cover, T. L.,
C. P. Dooley,
and
M. J. Blaser.
Characterization of and human serologic response to proteins in Helicobacter pylori broth culture supernatants with vacuolizing cytotoxin activity.
Infect. Immun.
58:
603-610,
1990[Medline].
6.
Cover, T. L.,
Y. Glupczynski,
A. P. Lage,
A. Burette,
M. K. Tummuru,
G. I. Perez-Perez,
and
M. J. Blaser.
Serologic detection of infection with CagA+ Helicobacter pylori strains.
J. Clin. Microbiol.
33:
1496-1500,
1995[Abstract].
7.
Cover, T. L.,
M. K. Tummuru,
P. Cao,
S. A. Thompson,
and
M. J. Blaser.
Divergence of genetic sequences for the vacuolating cytotoxin among Helicobacter pylori strains.
J. Biol. Chem.
269:
10566-10573,
1994
8.
Craig, P. M.,
M. C. Territo,
W. E. Karnes,
and
J. H. Walsh.
Helicobacter pylori secretes a chemotactic factor for monocytes and neutrophils.
Gut
33:
1020-1023,
1992[Abstract].
9.
Giraud, A. S.,
A. H. Soll,
F. Cuttitta,
and
J. H. Walsh.
Bombesin stimulation of gastrin release from canine gastrin cells in primary culture.
Am. J. Physiol.
252 (Gastrointest. Liver Physiol. 15):
G413-G420,
1987
10.
Graham, D. Y.,
M. F. Go,
G. M. Lew,
R. M. Genat,
and
J. F. Rehfeld.
Helicobacter pylori infection and exaggerated gastrin release. Effects of inflammation and progastrin processing.
Scand. J. Gastroenterol.
28:
690-694,
1993[Medline].
11.
Graham, D. Y.,
A. R. Opekun,
G. M. Lew,
D. J. Evans,
P. D. Klein,
and
D. G. Evans.
Ablation of exaggerated meal-stimulated gastrin release in duodenal ulcer patients after clearance of Helicobacter (Campylobacter) pylori infection.
Am. J. Gastroenterol.
85:
394-398,
1990[Medline].
12.
Graham, D. Y.,
A. Opekun,
G. M. Lew,
P. D. Klein,
and
J. H. Walsh.
Helicobacter pylori-associated exaggerated gastrin release in duodenal ulcer patients. The effect of bombesin infusion and urea ingestion.
Gastroenterology
100:
1571-1575,
1991[Medline].
13.
Harris, P. R.,
H. L. Mobley,
G. I. Perez-Perez,
M. J. Blaser,
and
P. Smith.
Helicobacter pylori urease is a potent stimulus of mononuclear phagocyte activation and inflammatory cytokine production.
Gastroenterology
111:
419-425,
1996[Medline].
14.
Langenberg, W.,
E. A. Rauws,
A. Widjojokusumo,
G. N. J. Tytgat,
and
H. C. Zanen.
Identification of Campylobacter pyloridis isolates by restriction endonuclease DNA analysis.
J. Clin. Microbiol.
24:
414-417,
1986[Medline].
15.
Lehmann, F. S.,
E. H. Golodner,
J. Calam,
M. Chen,
K. Kato,
and
A. H. Soll.
Cytokine and mononuclear cell stimulation of gastrin release from cultured canine antral G cells (Abstract).
Gastroenterology
108:
A860,
1995.
16.
Lehmann, F. S.,
E. H. Golodner,
J. Wang,
M. C. Chen,
D. Avedian,
J. Calam,
J. H. Walsh,
S. Dubinett,
and
A. H. Soll.
Mononuclear cells and cytokines stimulate gastrin release from canine antral cells in primary culture.
Am. J. Physiol.
270 (Gastrointest. Liver Physiol. 33):
G783-G788,
1996
17.
Leunk, R. D.,
P. T. Johnson,
B. C. David,
W. G. Kraft,
and
D. R. Morgan.
Cytotoxic activity in broth-culture filtrates of Campylobacter pylori.
J. Med. Microbiol.
26:
93-99,
1988[Abstract].
18.
Levi, S.,
K. Beardshall,
I. Swift,
W. Foulkes,
R. Playford,
P. Ghosh,
and
J. Calam.
Antral Helicobacter pylori, hypergastrinaemia and duodenal ulcers. Effect of eradicating the organism.
Br. Med. J.
299:
1504-1505,
1989[Medline].
19.
Levi, S.,
G. Haddad,
P. Ghosh,
K. Beardshall,
R. Playford,
and
J. Calam.
Campylobacter pylori and duodenal ulcers: the gastrin link.
Lancet
1:
1167-1168,
1989[Medline].
20.
Mai, U. E.,
G. I. Perez,
L. M. Wahl,
S. M. Wahl,
M. J. Blaser,
and
P. D. Smith.
Soluble surface proteins from Helicobacter pylori activate monocytes/macrophages by a lipopolysaccharide-independent mechanism.
J. Clin. Invest.
87:
894-900,
1991[Medline].
21.
Markwell, M. A. K.,
S. M. Haas,
L. L. Bieber,
and
N. E. Tolbert.
A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.
Anal. Biochem.
87:
206-210,
1978[Medline].
22.
Moss, S. F.,
and
J. Calam.
Acid secretion and sensitivity to gastrin in patients with duodenal ulcer: effect of eradication of Helicobacter pylori.
Gut
34:
888-892,
1993[Abstract].
23.
Moss, S. F.,
S. Legon,
A. Bishop,
J. Polak,
and
J. Calam.
Effect of Helicobacter pylori on gastric somatostatin in duodenal ulcer disease.
Lancet
340:
930-932,
1992[Medline].
24.
Phillips, E.,
and
P. Nash.
Culture media.
In: Manual of Clinical Microbiology (4th ed.), edited by E. H. Lennette,
A. Balows,
W. J. Hausler, Jr.,
and H. J. Shadomy. Washington, DC: Am. Soc. Microbiol., 1985, p. 1051-1092.
25.
Schepp, W.,
A. H. Soll,
and
J. H. Walsh.
Dual modulation by adenosine of gastrin release from canine G-cells in primary culture.
Am. J. Physiol.
259 (Gastrointest. Liver Physiol. 22):
G556-G563,
1990
26.
Weigert, N.,
K. Schaffer,
V. Schusdziarra,
M. Classen,
and
W. Schepp.
Gastrin secretion from primary cultures of rabbit antral G cells: stimulation by inflammatory cytokines.
Gastroenterology
110:
147-154,
1996[Medline].
27.
Yong, H. C.,
J. H. Walsh,
H. Yang,
Y. Taché,
and
A. M. Buchan.
A monoclonal antibody to somatostatin with potent in vivo immunoneutralizing activity.
Peptides
11:
707-712,
1990[Medline].
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