hypergastrinemia in response to gastric inflammation suppresses somatostatin

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Hypergastrinemia in response to gastric inflammation suppresses

somatostatin.

Yana Zavros1, Gabriele Rieder2, Amy Ferguson4, Linda C. Samuelson3 and Juanita L.

Merchant1,2,3

Howard Hughes Medical Institute1 and the Departments of Internal Medicine2,

Physiology3 and Pathology4 University of Michigan, Ann Arbor, MI

Running Title: Suppression of somatostatin by hypergastrinemia.

Corresponding Author

Juanita L. Merchant, M.D., Ph.D.

1150 West Medical Drive

MSRB I, 3510

Ann Arbor, MI 48109-0650

Phone: (734) 647-2944

Fax: (734) 936-1400

email: merchanj@umich.edu

Abbreviations: wild type mice (G+/+); gastrin-deficient mice (G-/-); G cells (gastrin-

expressing cells); D cells (somatostatin-expressing cells); vehicle treated mice (V);

omeprazole treated mice (OM); antibiotics (Abx); colony forming units (CFU).

Copyright 2001 by the American Physiological Society.

AJP-GI Articles in PresS. Published on October 15, 2001 as DOI 10.1152/ajpgi.00287.2001

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Key Words: Helicobacter pylori, bacterial overgrowth, omeprazole, gastrin-null mice,

mucosal lymphocytes.

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Abstract

Background: Hypergastrinemia and a reduction in tissue somatostatin occur in

Helicobacter pylori-infected patients. We investigated whether the D cell may be a direct

target of gastric inflammation and hypergastrinemia. Methods: D cells were quantified

by morphometry and flow cytometry in 16 week old wild type (G+/+) and gastrin-

deficient (G-/-) mice. Hypochlorhydric G-/- mice were treated with either antibiotics for

20 days or infused with gastrin (G-17) for 14 days. G+/+ mice were made

hypochlorhydric by treating mice with omeprazole for 2 months. Results: G-/- mice

showed significant inflammation when compared to the G+/+ mice which resolved after

20 days of antibiotic treatment. D cell numbers were not significantly different between

G-/- and G+/+ mice. After infusing G-17, fundic and antral D cell numbers decreased in

the G-/- mice. G+/+ animals made hypergastrinemic with omeprazole exhibited

decreased D cell numbers. When omeprazole treated mice were treated with antibiotics

alone, elevated plasma gastrin levels returned to baseline and D cell numbers returned to

resting levels despite persistent hypochlorhydria. Conclusion: Hypergastrinemia,

induced by inflammation, results in decreased D cell numbers. Thus, the stomach

responds to the presence of inflammation by reducing somatostatin levels thereby

releasing the inhibition on the G and parietal cells to maximize gastric acid output.

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Introduction

It has been established that Helicobacter pylori (H. pylori) induces mucosal

inflammation or gastritis and subsequently major cellular and physiologic changes in the

stomach (10). In particular, H. pylori colonization of the stomach correlates with an

increase in gastric G cells, gastrin expression and secretion (10). Basal levels of acid

secretion rise, presumably due to the elevated levels of circulating gastrin. It is assumed

that the high gastric acid levels stimulate the production of somatostatin through putative

chemoreceptors on the D cell. Once the D cell is activated, secreted somatostatin should

inhibit the G cell by paracrine mechanisms (23). Rather, what is observed in H. pylori-

infected patients are reduced numbers of D cells and somatostatin in the face of elevated

gastrin production and acid secretion (12, 24). To explain these findings, a direct

inhibitory effect by inflammatory cytokines on D cells has been proposed (2, 3, 5).

Evidence for the immunoregulation of somatostatin originates from in vitro studies

demonstrating that cytokines such as TNFα and IL-8 inhibit somatostatin release from

isolated rabbit and canine fundic D cells [(2, Beales, 1998 #6168)].

Gastrin null mice created by homologous recombination are hypochlorhydric (11,

13). We have recently studied these mice in greater detail and have found that they also

exhibit significant gastric mucosal inflammation caused by bacterial overgrowth as a

consequence of reduced gastric acidity (30). The present study extends these findings by

measuring changes in tissue somatostatin and D cell numbers in gastrin null mice. As

reviewed above, the levels of tissue somatostatin and the D cell numbers are quite

relevant to understanding the regulation of gastric acid secretion. Thus, it is important to

evaluate the reason for suppressed tissue somatostatin levels during gastritis. Our studies

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in the gastrin-deficient and omeprazole-treated mice indicate that in the presence of

gastritis, the stomach is stimulated to maximize acid output (30). Thus, it is logical to

propose that at the same time the stomach will act to suppress the inhibitors of gastric

acid secretion. Since somatostatin is a major inhibitor of gastrin secretion and therefore

acid secretion, we hypothesize that during inflammation somatostatin production is

actively inhibited.

Analyzing the D cell population in the gastrin-deficient mice, we observed and

report here that both D cells and somatostatin levels remain elevated despite significant

gastritis. This raised the interesting possibility that gastrin may be the regulator of the D

cell. We tested this hypothesis by infusing gastrin into the gastrin-deficient mice in the

presence of antibiotics to resolve the inflammation. We found that the gastrin infusion

suppressed both fundic and antral D cell populations and tissue somatostatin levels. We

also examined changes in D cells in mice made hypergastrinemic by omeprazole.

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Material and Methods

Animals: Gastrin deficient (G-/-) mice and strain-matched (C57Bl/6 x

129/Sv) wild type controls (G+/+) were bred by homozygous mating. G+/+ (n=10) and

G-/- (n=12) mice were maintained in individual, sterile microisolator cages in non-barrier

mouse rooms for 16 weeks. All mice were fasted overnight with access to water ad

libitum before analysis. The study was performed with the approval of the University of

Michigan Animal Care and Use Committee which maintains an American Association of

Assessment and Accreditation of Laboratory Animal Care (AAALAC) facility.

Antibiotic treatment: At 13 weeks of age G+/+ and G-/- were treated with

antibiotics by adding 5 mg/kg of streptomycin to their drinking water for 3 weeks. At the

same time, mice were given a subcutaneous injection of 100 mg/kg/day of cefoperazone

(Sigma) (26). Each day their feces were collected, weighed, suspended in 1 ml PBS by

vortexing, then diluted 1:100. One hundred microliters of the suspension were cultured

on Luria broth plates and incubated at 37oC for 24 hr. The colonies on each plate were

counted and the results expressed as CFU/g feces. After 5 days of antibiotic treatment,

the CFU/g feces decreased significantly from 300 x 103 CFU/g feces to 85 x 103 CFU/g

feces. The bacterial count continued to decrease over the next 15 days of treatment (data

not shown) and bacterial counts were undetected in mouse feces by day 20. Therefore we

chose this duration for treatment. Mice were sacrificed after 20 days of antibiotic

treatment and analyzed.

G-17 infusion: G-/- mice were anaesthetized with xyalzine (20 mg/ml) and

ketamine (100 mg/ml) at 8 weeks of age. A midline incision was made in the skin below

the rib cage. Another incision was then made in the abdominal muscle directly under the

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cutaneous incision. The micro-osmotic pump (Alzet, model 1002), delivering 5µg/kg/hr

of rat non-sulfated gastrin (G-17) (Bachem) was inserted into the peritoneal cavity. The

muscle and skin incisions were then closed by silk sutures. Mice were continuously

infused with G-17 for 14 days prior to sacrifice and analysis.

Omeprazole treatment: Eight week old G+/+ mice were made

hypochlorhydric by treating with a single intraperitoneal injection of omeprazole per day

(400 µmol/kg) for 8 weeks. The stock solution of omeprazole (80 µmol/ml) was

dissolved in DMSO/PEG (4.5/0.5 vol/vol) and stored at –20oC until use. Eight week old

control mice were treated in the same manner with the vehicle alone. All mice were

sacrificed at 16 weeks of age and analyzed. In a separate group, 8 week old G+/+

animals were also treated with omeprazole for 8 weeks, but were treated with the same

antibiotic regimen as described above for 20 days. These animals were also sacrificed at

16 weeks of age.

Quantitative and Qualitative Microbiology: The wet weight of the antral

and fundic tissue was determined. Tissue was homogenized briefly with a polytron PT-

2000 (Kinematica, Cincinnati, OH) in 1 ml saline, and appropriate dilutions were spread

on blood agar plates containing Campylobacter Base Agar (Difco, MI) supplemented

with 5% horse blood (Colorado Serum, Denver) and 2 µg/ml nystatin (Sigma, MO).

Plates were incubated under aerobic, microaerophilic and anaerobic conditions

(CampyPak Plus, GasPak Plus, Anaerobic Systems, BBL) at 37oC for 3-5 days. The

number of colonies were counted and the colony forming units (CFU) per gram of tissue

calculated. Comparisons between groups were based on the log concentrations of

bacteria. For statistical analysis, the total viable count was determined by a total colony

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count of all bacterial types on aerobic, facultative anaerobic and anaerobic cultures.

Single colonies were used to identify major bacterial species, by the Medical

Microbiology Department at the University of Michigan Hospital, using specific nutrient

culture conditions, biochemical assays and API System for fermenting and non-

fermenting Gram negative bacteria.

To exclude the presence of Helicobacter pylori (H. pylori), an aliquot of the

homogenate was spread on selective blood plates supplemented with 5 µg/ml

vancomycin and 10 µg/ml trimethoprim lactate (Sigma, MO), then incubated for 5 days

at 37oC under microaerophilic conditions. Single colonies from these plates were tested

for urease (using a drop of urea broth:10g urea, 0.5% w/v phenol red, 0.22 g

NaH2PO4.H2O, 0.51 g Na2HPO4, 100 mg NaN3, and 500 ml distilled water at pH 6.2),

catalase (using 3% H2O2 solution), and oxidase (DrySlide, BBL) activity. None of the

tested colonies were positive for any of the three enzyme tests. The quantitative urease

activity was performed on the remaining homogenate to exclude other urease positive

Helicobacter species. Briefly, homogenate was centrifuged for 10 min at 7500 rpm and

the pellet resuspend in 200 µl of urea broth containing phenol red. After incubation at

37oC over-night, the reaction mixture was centrifuged and the supernatants were placed

in microtiter plates. The extent of the phenol red color change was recorded in an

automated ELISA reader at 550 nm against a H. pylori standard curve. All colonies

tested were below the standard curve for H. pylori suggesting an absence of urease

positive Helicobacter spp. In addition, DNA was extracted from stomach tissue of G-/-

and G+/+ mice and a pure culture of H. pylori using a DNeasy Tissue Kit (Qiagen)

according to the manufacturers protocol. Helicobacter primer pairs C97 and C98 and

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C97 and C05 were used to amplify 16S rRNA amplicons of approximately 400 and 1200

base pairs respectively. The primer sequences were generated according to Fox et al.

(10) and were as follows: C97, 5’-GCT ATG ACG GGT ATC C-3’ (276-291 forward);

C98, 5’-GAT TTT ACC CCT ACA CCA-3’(681-698 reverse) and C05, 5’-ACT TCA

CCC CAG TCG CTG-3’ (1478-1494 reverse). PCR conditions are as previously

published (10). Analysis of the H. pylori DNA samples by PCR with the Helicobacter

genus-specific primers produced the expected 400 base pair and 1200 base pair

amplicons with H. pylori DNA extracted from the stomachs of G+/+ mice infected with

the SS1 strain (16). Neither of the 400 and 1200 base pair amplicons was amplified from

DNA extracted from uninfected G+/+ or G-/- mice, confirming the culture results

demonstrating that there were no Helicobacter spp. present in these mice.

Immunohistochemistry: A longitudinal section of the stomach (spanning

both the fundic and antral regions) was fixed in 4% paraformaldehyde/PBS, paraffin-

embedded and 3 µm sections were prepared. Sections were deparaffinized then

permeabilized in 3% H2O2 and 100% ethanol. Non-specific antigenic sites were blocked

with 20% normal goat serum/PBS, 0.1% Triton X-100 for 30 min before a 2 h incubation

with a 1:200 dilution of rabbit anti-somatostatin (Zymed) antibody. A 1:500 dilution of

the secondary anti-rabbit IgG antibody was added for 30 min and visualized with avidin-

biotin complexes using the Vectastain Elite ABC Kit and diaminobenzidine (DAB) for

substrate (Vector Laboratories, Inc., Burlingame, CA). Sections were also stained with

hematoxylin and eosin (H&E). The morphometric results were expressed as the average

number of cells counted per gland. A total of 10 oriented glands in random fields were

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counted for each mouse. The results were expressed as the average number of cells

counted per gland.

Sections stained by H&E were graded on the intensity of inflammation and

metaplasia by a pathologist blinded to the treatment and mouse genotypes according to

Eaton et al, a grading system developed for the histologic quantification of gastritis in

mice (9). A score of 0-1 was given to those sections showing no inflammation, 2 for

gastritis (inflammatory infiltrate sufficient to displace glands and 3 for marked

inflammation with metaplasia, where metaplasia is defined as the loss of normal fundic

morphology with replacement of mucous-secreting glands (9). The presence of

metaplasia was confirmed by a PAS/alcian blue stain.

Blood collection: After sacrifice, approximately 1 ml of blood was collected

by cardiac puncture, aliquoted into tubes that were lithium heparinized and centrifuged at

15,000 rpm for 15 min at 4oC. Plasma was collected immediately and stored at –20oC

until assayed.

Peptide extraction: Gastric sections were weighed and added to 500 µl of

boiling double distilled water. After boiling for 5 min the biopsies were compressed

using a glass rod, the tissue was boiled for an additional 5 min, then microfuged at 10,000

rpm for 15 min. The supernatant collected was designated as the water extract containing

gastrin. The pellet was resuspended in 500 µl of boiling 3% acetic acid, boiled for 10

min and microfuged as above. The supernatant collected was the acid extract containing

somatostatin. Extracts were stored at –20oC until assayed by radioimmunoassay.

Somatostatin radioimmunoassay: Somatostatin concentrations in water and

acid biopsy extracts were measured using appropriate volumes and dilutions. Antiserum

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1001 (CURE, UCLA) was used, which detects both somaotstatin-14 and somatostatin-28

(18). 125I-Tyr-somatostatin-14 was used as the label and somatostatin-14 (50 pmol/L)

was used to generate the standard curve. The ID50 was 8 fmol/ml and the inter- and intra-

assay coefficients of variation were less than 5% and 12% respectively.

Gastrin radioimmunoassay: Plasma and the water and acid tissue extracts were

assayed for gastrin amide (G-17) using appropriate volumes and dilutions (empirically

determined). Samples were incubated in duplicate at 4oC with 125I-15Met human G-17

label and antiserum 1296 (CURE, UCLA). Antiserum 1296 recognizes all carboxy-

terminal fragments larger than the pentapeptide and measures G-17 sulfated and non-

sulfated identically. G-17 (50 pmol/L) was used to generate the standard curve. The ID50

was 1 fmol/ml and inter- and intra-assay coefficients of variation were less than 2% and

11% respectively.

Gastric acid concentrations: The stomachs from G-/- mice infused with

G-17 were opened along the greater curvature and washed with 2 ml normal saline (pH

7.0). All mice were fasted overnight before gastric samples were collected. The

contents were centrifuged at 3,000 rpm for 5 min and the supernatant collected. The

supernatant was titrated using 0.005N NaOH and gastric acidity was expressed in µEq.

Cell preparation and flow cytometry: Lymphocytes and epithelial cells

were isolated from the gastric mucosa according to a previous method (7). Briefly, the

stomach was dissected into 2 mm size pieces. The pieces were first incubated in 20 ml of

Hank's balanced salt solution containing 5% BSA, 1 mM DTT and 1 mM EDTA for 1 h

with vigorous shaking at 37oC to release the epithelial cell population. This first cell

suspension was passed through a filter (50 µm Filcon filter, Dako), collected and washed

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twice with RPMI medium containing 5% fetal calf serum. The stripped mucosa was then

subjected to enzymatic digestion in 20 ml of RPMI medium containing 1 mg/ml dispase

II (Roche Molecular Biochemicals) for two 30 min incubations at 37oC with vigorous

shaking to remove mucosal lymphocytes. The lymphocytes were collected, washed and

surface labeled for flow cytometry. Isolated T cells, B cells and leukocytes were labeled

with FITC-conjugated anti-mouse CD3 (Pharmingen), CD19 (Pharmingen) and

phycoerythrin-conjugated anti-mouse CD45 (Pharmingen) respectively. D cells were

labeled with a rabbit anti-somatostatin (Vector Labs.) antibody after permeabilization

(29). The total epithelial cell population was quantified using a monoclonal anti-human

cytokeratin-18 (keratin RCK106) antibody (catalogue number 11416, Cappel ICN

Pharmaceuticals). FITC-conjugated anti-mouse and anti-rabbit immunoglobulins

(Cappel ICN Pharmaceuticals) were used to detect the cytokeratin-18 or somatostatin

primary antibodies respectively. Labeled cells were then analyzed by flow cytometry

using a Coulter Elite ESP Cell Sorter (Bechman-Coulter Electronics, Florida). A total of

10,000 cells were analyzed for all cell types. Changes in T and B cells were calculated as

follows:

Cell number in total cell prep x % CD45 + cells x % CD3+ or CD19 + cells

= Number of T or B cells in the mucosa (1). D cells were expressed as a percentage of

the number of cytokeratin-18 positive cells.

Statistical analysis: The results were statistically tested by unpaired t-test or

one-way ANOVA as appropriate, using commercially available software (GraphPad

Prism, GraphPad Software, San Diego, CA). A P<0.05 was considered significant.

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Results

Mucosal inflammation in gastrin-deficient (G-/-) mice. We examined

mucosal changes by histology and by flow cytometry to quantify T and B cell

populations. Significant inflammation was observed in G-/- (Fig. 1B) compared to the

G+/+ mice (Fig. 1A). The histologic grades for all mice were scored by a pathologist

blinded to the experiment. The mean histologic score for G-/- mice was 3.58 + 0.38

which was significantly greater than that of the G+/+ mice (0.75 + 0.13). To determine if

the presence of bacteria was the cause of the inflammation in the G-/- mice, a group was

treated with antibiotics for 20 days. After antibiotic treatment, the histologic grade

scored in the G-/- mice decreased to normal (0.50 + 0.15). Although the histologic scores

decreased after G-/- mice were infused with G-17 (1.42 + 0.19), this grade was

significantly greater than that for the G+/+ animals. To support the qualitative analysis of

mucosal inflammation, flow cytometry was used to quantify changes in the number of T

and B lymphocytes. Consistent with the histologic grading, there was a significant

increase in T and B cells in G-/- mice that resolved with antibiotics. As observed in the

histologic analysis, G-17 infusion reduced the T and B cell populations (Fig. 2).

However, the lymphocyte population after G-17 infusion did not return completely to

baseline levels.

G-17 infusion reduced bacterial overgrowth in the G-/- mice. Figure 3

illustrates the total number of bacterial counts in the G+/+ mice versus G-/- mice in both

the fundus (Fig. 3A) and antrum (Fig. 3B). After antibiotic treatment and G-17 infusion,

there was a significant reduction in the total number of bacteria in both the fundus and

antrum. However, these numbers were significantly greater than the bacterial counts for

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the G+/+ mice treated with antibiotics. Overall, the total number of bacteria in the

antrum was significantly greater in the G-/- mice compared to the G+/+ animals.

D cells and tissue somatostatin were suppressed after G-17 infusion. To

evaluate the response to inflammation, changes in D cells and tissue somatostatin

concentrations were measured in G-/- mice. Immunohistochemical staining revealed

positive anti-somatostatin antibodies in both the fundus and antrum of the G-/- mice (Fig.

4A and B). These cells were quantified by morphometric analysis and showed that, after

antibiotic treatment there was no change in the number of D cells in either the fundus or

antrum (Fig. 5B). In contrast to the antibiotic treatment, after G-17 infusion there was a

reduction in the number of D cell staining in both the fundus and antrum (Fig. 4C and D).

After G-17 infusion, there was a significant decrease in D cell numbers in both fundic

and antral regions of the stomach (Fig. 5B). In addition, there was a significant reduction

in both fundic and antral tissue somatostatin concentrations after G-17 infusion (Table 1).

Thus, hypergastrinemia had a greater suppressive effect than inflammation on the D cell

numbers and somatostatin levels. The decrease in somatostatin was consistent with an

increase in circulating gastrin (558 + 114 pmol/l) in the G-/- mice infused with G-17

compared to resting levels in the G+/+ mice (54 + 8 pmol/l) (Table 1). The reduction in

somatostatin during G-17 infusion also correlated with an increase in gastric acidity.

Gastric acidity increased from 0.48 + 0.10 µEq in the untreated G-/- mice to 1.86 + 0.36

µEq in the G-17 infused mice. However, gastric acidity did not reach acid levels

measured in the G+/+ mice (2.71 + 0.31 µEq) (Table 1). This result was similar to the

incomplete recovery of acid secretion observed by Friis-Hansen et al (11). There was no

change in gastric acidity in G-/- mice after antibiotic treatment. Flow cytometry, which

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evaluated the total number of D cells in the epithelium correlated with the morphometric

analysis (Fig. 5A). Both morphometric and flow cytometric analyses revealed that there

was a trend toward lower D cell numbers in the G-/- mice. This may reflect the modest

inhibitory effect of inflammation on the D cell. Slightly fewer D cells were counted in

the antrum compared to the fundus although there were 3-fold higher tissue somatostatin

levels in the antrum compared to the fundus. Therefore, it appears that the concentration

of somatostatin per cell is highest in the antrum. In addition, there was no significant

difference in somatostatin concentrations between G+/+ and G-/- mice (Table 1).

Hypergastrinemia in omeprazole-treated G+/+ mice suppressed D cells and

tissue somatostatin. Collectively, our results suggest that somatostatin is suppressed by

increasing circulating gastrin concentrations. Furthermore, hypergastrinemia induced by

omeprazole treatment is also associated with decreased somatostatin. Therefore, to

further evaluate the role of hypergastrinemia in the down-regulation of somatostatin,

G+/+ mice were made hypochlorhydric and hypergastrinemic with omeprazole treatment

for 2 months. The circulating gastrin concentrations in these mice were 159 + 11 pmol/l

(Table 2), as reported in other animal models and human subjects (7). Histologic and

flow cytometric evaluation revealed that G+/+ omeprazole-treated mice also showed

significant inflammation (30). After 2 months of omeprazole treatment, D cell numbers

were significantly decreased in both the fundus and antrum compared to untreated

animals (Fig. 6B). This correlated with a significant reduction in tissue somatostatin

concentrations in both the fundus and antrum in the omeprazole-treated mice compared to

controls (Table 2). After antibiotic treatment, the elevated circulating gastrin

concentrations returned to resting levels (34 + 4 pmol/l). Subsequently, we found that

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resolution of the inflammation correlated with a return of tissue somatostatin levels

(Table 2) and D cell numbers to baseline (Fig. 6B). The flow cytometry was consistent

with the morphometry (Fig. 6A). The return of the D cells to baseline levels occurred in

the presence of hypochlorhydria documented by acid concentrations of 1.62 + 0.59 µEq

in omeprazole-treated mice compared to 5.62 + 0.70 µEq in untreated animals (Table 2).

Figure 7 illustrates the differences between the G-/- and omeprazole-treated

mouse models. G+/+ mice treated with omeprazole become hypochlorhydric and thus

are susceptible to bacterial overgrowth and inflammation. A consequence of the

inflammation is the increase in plasma gastrin (30) (Fig. 7A). When these mice were

treated with antibiotics and the inflammation resolved, plasma gastrin concentrations

normalized despite the stomach remaining hypochlorhydric (Fig. 7B, Table 2).

Examining the fate of the D cell population, we found that D cell numbers were

depressed in the hypergastrinemic state induced by omeprazole (G+/+ mice) (Fig. 6).

Certainly, this is as expected from prior studies suggesting that low acid levels inhibit the

D cell (23). However, we challenged this dogma by administering antibiotics to the

omeprazole-treated mice to eliminate the inflammation-induced hypergastrinemia. With

the reduction in plasma gastrin levels, the D cells returned to baseline resting levels

despite persistent hypochlorydria (Fig. 7B, Table 2). Therefore, we concluded that D

cells are regulated by fluctuations in gastrin and not gastric acid which did not change.

The results in the omeprazole model are supported by studies using G-/- mice. G-

/- mice are hypochlorhydric due to a genetic deficiency of gastrin. These mice exhibit

significant inflammation and increased G cell density (30) despite a lack of gastrin.

Since D cell numbers and somatostatin concentrations were unchanged even after

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antibiotic treatment, this lead us to propose that gastrin may be the critical factor required

to suppress somatostatin production (Fig. 7C). Confirming our suspicions, it was only

after the infusion of G-17 into the gastrin null mice that we observed a reduction in

somatostatin and D cell density (Fig. 7D). Therefore, these results clearly show that

hypergastrinemia is the major factor modulating the D cell during bacterial infection.

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Discussion

We have shown that hypochlorhydria, induced either genetically by deletion of

the gastrin gene, or chemically by treatment with omeprazole, causes bacterial

overgrowth that results in gastric inflammation (30). There were slightly lower D cell

numbers in G-/- mice which may reflect an effect of chronic inflammation on

somatostatin in the G-/- mice. While antibiotic treatment of G-/- mice resolved the

inflammation and normalized cell populations such as parietal and G cells (30), we show

here that there was no significant effect on D cells or tissue somatostatin content. When

G-/- mice were infused with G-17, raising circulating gastrin concentrations to levels

observed in hypergastrinemic animals, D cell numbers and tissue somatostatin levels

were suppressed. Similarly, hypergastrinemic omeprazole-treated G+/+ mice also

exhibited reduced D cell numbers and tissue somatostatin concentrations. After

omeprazole mice were treated with antibiotics, plasma gastrin concentrations normalized,

correlating with resolution of the inflammation. Consistent with lower circulating gastrin

levels, D cell numbers returned to baseline. This was also observed in the omeprazole-

treated mice despite persistent hypochlorhydria. Therefore, we conclude from these

studies that the suppression of somatostatin during bacterial infection is due to the

hypergastrinemia induced in response to inflammation and not the hydrogen ion

concentration as previously proposed.

In wild type mice and human subjects, omeprazole induces significant

hypergastrinemia presumably due to the decrease in gastric acid that in turn inhibits D

cell secretion of somatostatin (14, 15). Moreover, in the absence of somatostatin,

inhibition of the G cell is removed allowing serum gastrin levels to rise (6, 21, 22). Since

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we have shown that omeprazole treatment induces a gastritis and that the inflammation is

a major activator of the G and parietal cell, our model demonstrates that G cell numbers

should increase and serum gastrin levels should rise (30). Indeed, this is what we

observed and is also what has been observed in other models of chemical achlorhydria in

rodents and in human subjects (4, 17, 28). In these prior reports, modulation of the G and

D cell population was postulated to be due to changes in gastric acid. Our model would

support the notion that the regulation of gastrin and somatostatin by gastric acid is

indirect. Rather, the absence of acid permits bacterial overgrowth or microbial

metabolites to induce inflammation. Consistent with this hypothesis, we found that in

normal mice treated with omeprazole and antibiotics to resolve the inflammation, gastrin

levels decreased and D cells returned to their normal resting numbers despite low gastric

acidity. Thus, in the face of low gastric acid due to omeprazole, gastrin levels were

suppressed simply by treating the animals with antibiotics. This in turn released the

inhibition on the D cell. If hypoacidity were the major direct regulator as previously

proposed, then the D cell numbers and tissue somatostatin should have remained low.

Although prior studies have indicated that gastrin stimulates somatostatin release, these

studies were performed on isolated cell populations or in whole animals in the absence of

inflammation, hypochlorhydria or bacterial overgrowth (8, 31). In addition, these studies

examined the effect of acutely administering gastrin. Thus, we emphasize that the results

shown here document an inhibitory effect of gastrin on somatostatin secretion in vivo

when hypergastrinemia is induced by chronic inflammation. Since the studies were

performed in vivo, it is not known under these conditions whether gastrin stimulates the

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release of other ligands, e.g., HB-EGF, TGFα or trefoil proteins, or acts directly on the D

cell (19, 20, 25, 27). Certainly both possibilities may also occur in concert.

In summary, the findings reported here clearly show that hypergastrinemia,

induced by inflammation, results in decreased D cell numbers. The stomach responds to

the presence of inflammation by increasing gastric acidity, elevating G cell numbers and

gastrin secretion that in turn stimulates parietal cells (30). Coincident with the changes to

increase acid secretion, hypergastrinemia also blocks the somatostatin inhibitory pathway

(Fig. 8). As a result, the stomach is able to maximize its gastric acid output in an attempt

to clear bacterial infection.

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Acknowledgements

Y.Z. is an associate and J.L.M. is an assistant investigator of the Howard Hughes Medical

Institute. This work was supported in part by Public Health Service grant DK-45729

(J.L.M.) and DK48815 (LCS). We acknowledge the assistance of the flow cytometry core

in the University of Michigan Cancer Center (CA46952) and University of Michigan

Multipurpose Arthritic Center (AR20557) and the assistance of the radioligand core of

the University of Michigan Gastrointestinal Peptide Research Center (DK-34533). We

also acknowledge the use of rabbit gastrin and somatostatin antibodies #1296 and #1001

respectively, from CURE DDRC/RIA Core (DK41301). We thank Chris Dickinson for

synthesizing iodinated somatostatin peptide. We thank Judy Poore for her assistance

with the immunohistochemistry and Mary Van Antwerp and Arthur Tessier for their

technical assistance.

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Figure Legends

Figure 1: Representative H&E stains from mouse stomach. Representative H&E

stained sections of inflamed stomach from a 16 week old (A) G+/+, (B) G-

/-, (C) G-/- mouse treated with antibiotics or (D) G-/- mouse infused with

G-17. The arrows indicate areas of inflammation. Histologic scores

(mean + SEM) are shown in parentheses. n=10 for the G+/+ group, n=12

for the G-/- groups.

Figure 2: Analysis of T and B lymphocytes by flow cytometry. T and B

lymphocytes isolated from the stomach of G+/+ (open bars), G+/+ mice

treated with antibiotics (shaded bars), G-/- mice (closed bars), G-/- mice

treated with antibiotics (stippled bars) and G-/- mice infused with G-17

(hatched bars) were analyzed by flow cytometry and expressed as the

percent of total cell numbers in prep. *P<0.05 versus G+/+ mice, n=10 for

G+/+ group and n=12 for G-/- group.

Figure 3: Bacterial counts from the stomachs of G+/+ and G-/- mice treated

with either antibiotics or G-17 infusion. Total bacterial counts in the

mouse (A) fundus and (B) antrum represented as the mean + SEM of

CFU/g tissue. Note numbers for G+/+ and G-/- mice treated with

antibiotics previously reported (30).

23

Figure 4: Distribution of D cells in the stomach. D cell distribution in G-/- mouse

(A) fundus, (B) antrum, (C) fundus infused with G-17, (D) antrum infused

with G-17 ad (E) high power of D cell showing cytoplasmic process

connecting with neighboring cell.

Figure 5: Quantitation of D cells. (A) By flow cytometry, the number of

somatostatin positive cells was determined by flow cytometry and is

shown for G+/+ mice (open bars), G+/+ mice with antibiotics (shaded

bars), G-/- mice (closed bars), G-/- mice treated with antibiotics (stippled

bars) and G-/- mice infused with G-17 (hatched bars). Somatostatin

positive cells were normalized to cytokeratin-18 staining to determine %

of D cells in the mucosa. (B) By morphometry, the number of D

cells/gland in G+/+ mice (open bars), G+/+ antibiotic treated mice (shaded

bars), G-/- mice (closed bars), G-/- treated with antibiotics (stippled bars)

and G-/- mice infused with G-17 (hatched bars) were counted. *P<0.05

versus G+/+ mice, n=10 for G+/+ group, n=12 for G-/- group.

Figure 6: Quantitation of D cells . (A) The percent of anti-somatostatin positive

cells per cytokeratin-18 positive cells was determined by flow cytometry

and is shown for controls (open bars), controls plus antibiotics (closed

bars), omeprazole (stippled bars) and omeprazole plus antibiotic (hatched

bars) treated G+/+ mice. (B) The number of D cells/gland in controls

(open bars), controls plus antibiotics (closed bars), omeprazole (stippled

24

bars) and omeprazole plus antibiotic (hatched bars) treated G+/+ mice

were counted. *P<0.05 versus controls, n=8.

Figure 7: Comparison between omeprazole (PPI) treated and G-/- mouse

models. (A) hypergastrinemia during PPI treatment of G+/+ mice results

in elevation of D cell density, (B) after G+/+ mice are treated with

antibiotics plasma gastrin concentrations are normalized and D cells return

to baseline, (C) in the G-/- mice even after antibiotic treatment D cell

numbers are unaltered due to the absence of gastrin, however (D) G-/-

infused with gastrin-17 results in reduction in D cell numbers. Abx:

antibiotic treatment, *models that are comparable.

Figure 8: A proposed model for the down-regulation of somatostatin during

hypergastrinemia as a consequence of inflammation in (A) fundus and

(B) antrum. Inflammation will produce pro-inflammatory cytokines

which in turn may have an inhibitory effect on the D cell and an activating

effect of the G and parietal cells. Moreover, the increase in gastrin has a

direct or indirect inhibitory effect on the D cell. Thus, the overall effect

would be to maximize the acid output in response to gastric bacterial

colonization and inflammation.

25

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30

Table 1: Tissue somatostatin concentrations in the gastric mucosa.

Treatment Tissue somatostatin (pmol/g) Circulating gastrin

(pmol/l)#

Gastric acid

( Eq)#

Fundus Antrum

G+/+

AB

339 + 114

538 + 143

1272 + 301

1608 + 348

54 + 8 2.71 + 0.31

G-/-

AB

G-17

368 + 51

280 + 91

106 + 10*

1156 + 180

1225 + 237

497 + 98*

<2

558 + 114

0.48 + 0.10

1.86 + 0.36

*P<0.05 versus G+/+ group, # previously reported in (30)

Table 2: Tissue somatostatin concentrations in G+/+ omeprazole-treated mice.

Treatment Tissue somatostatin (pmol/g) Circulating gastrin

(pmol/l)#

Gastric acid

( Eq)#

Fundus Antrum

G+/+ V 212 + 30 1783 + 133 34 + 4 5.62 + 0.70

G+/+ V+AB 235 + 42 1621 + 119 44 + 5 5.03 + 0.66

G+/+ OM 186 + 22* 507 + 139* 159 + 11 1.62 + 0.59

G+/+ OM+AB 288 + 76 1496 + 65* 52 + 16 1.90 + 0.86

V: vehicle, AB: antibiotics, OM: omeprazole, *P<0.05 versus G+/+ control group, #

previously reported in (30)