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Published as doi: 10.1096/fj.05-5696fje.
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(The FASEB Journal. 2006;20:2396-2398.)
© 2006 FASEB

Gastrin induces leukocyte-endothelial cell interactions in vivo and contributes to the inflammation caused by Helicobacter pylori

Ángeles Álvarez*, Sales Ibiza{dagger}, Carlos Hernández*, Alberto Álvarez-Barrientos{ddagger}, Juan V. Esplugues*,1 and Sara Calatayud*

* Departamento de Farmacología and

{dagger} Unidad Mixta CNIC-UVEG, Facultad de Medicina, Universidad de Valencia, Valencia, Spain; and

{ddagger} Cytometry Unit, Fundación Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain

1Correspondence: Departamento de Farmacología, Facultad de Medicina, Universidad de Valencia, Avd. Blasco Ibáñez 15, 46010-Valencia, Spain. E-mail: juan.v.esplugues{at}uv.es

ABSTRACT

Gastric mucosal inflammation causes hypergastrinemia, and gastrin receptors have been detected in several leukocyte types. We have analyzed whether gastrin affects the leukocyte-endothelial cell interactions in vivo by monitoring leukocyte rolling, adhesion, and emigration in rat mesenteric venules using intravital microscopy. Mesenteric superfusion with exogenous gastrin increased these processes in a concentration- and time-dependent manner, effects prevented by the cholecystokinin (CCK)-2 receptor antagonists (proglumide, L-365,260) but not by the CCK-1 receptor antagonist devazepide. A similar response was induced by exogenous CCK or endogenously released gastrin. CCK-2 receptors were localized in mesenteric macrophages and polymorphonuclear leukocytes. This effect of gastrin is not modulated by somatostatin and is independent of the endogenous release of histamine. To analyze whether hypergastrinemia elicited by Helicobacter pylori (HP) modulates the inflammation induced by the germ, rats were chronically administered with an extract of a CagA+/VacA+ strain of HP. This protocol increased gastrinemia and induced an inflammatory response in the rat mesentery. Blockade of CCK-2 receptors attenuated this response and induced a qualitative change in the leukocyte infiltrate suggestive of a receding inflammatory process. Our results reveal a new proinflammatory role of gastrin that seems to contribute to the maintenance of the inflammation elicited by HP components.—Álvarez, A., Ibiza, S., Hernández, C., Álvarez-Barrientos, A., Esplugues, J. V., Sara Calatayud, S. Gastrin induces leukocyte-endothelial cell interactions in vivo and contributes to the inflammation caused by Helicobacter pylori.


Key Words: CCK-2 receptors • gastrin-releasing peptide

GASTRIN IS MAINLY KNOWN by its first described role as a gastric acid-secreting hormone and, more recently, by its ability to promote cellular growth. Peptides of the gastrin family activate two different receptors: the CCK-1 receptor, which has low affinity for gastrin but high affinity for the related hormone cholecystokinin (CCK), and the CCK-2 receptor, which has high affinity for both gastrin and CCK and mediates the acid-secretory as well as the proliferative effects of gastrin (1) . Recent studies using gene arrays have started to identify several previously unsuspected target genes downstream of the CCK-2 receptor, and accumulating evidence suggests that gastrin may have a wider range of effects (2) .

Gastrin receptors have been observed in several human leukocyte types. For instance, CCK-1 and CCK-2 receptors or their mRNAs have been localized in lamina propria macrophages (3) , peripheral blood mononuclear cells (4 , 5) , circulating polymorphonuclear (PMN) leukocytes (6) , and PMNs within the tumor stroma of human colorectal cancers (7) . In rats, pulmonary vascular endothelial cells and macrophages have been shown to express both CCK-1 and CCK-2 receptor genes (8) . In parallel with these molecular studies, gastrin and CCK have been shown to induce changes in leukocyte functions such as chemotaxis, adherence, or phagocitosis in vitro. In particular, both peptides seem to be chemoattractant for human neutrophils (9) , monocytes (10) , and lymphocytes (11) . However, it has not been analyzed whether these cellular effects of gastrin have any consequence on the functioning of the immune system in vivo.

It is important to note that infection by Helicobacter pylori (HP), the main cause of nonautoimmune chronic gastritis, increases gastrin secretion (12 13 14 15) , and a correlation between gastrinemia and the severity of gastritis has been identified (16 17 18) . This hypergastrinemia has been regarded as a consequence of the local inflammatory process, since it was shown that proinflammatory cytokines and activated monocytes stimulate gastrin release from antral G cells (19 20 21) . However, if gastrin could modify leukocyte behavior in vivo, its increased concentration in the HP-infected gastric mucosa might affect the evolution of the inflammatory process induced primarily by the microorganism, i.e., gastritis. In the present study, we have evaluated this possibility in rats administered chronically with an extract of a CagA+/VacA+ strain of HP, which mimics the HP infection in humans.

MATERIALS AND METHODS

Intravital microscopy
Leukocyte-endothelial cell interactions were evaluated in fasted male Sprague-Dawley rats (200–250 g), the details of the experimental preparation having been described previously (22 , 23) . In brief, rats were anesthetized with sodium pentobarbital (65 mg/kg, i.p.). A midline abdominal incision was made, and a segment of the midjejunum was exteriorized and placed over a transparent pedestal for tissue transillumination. A selected loop of the exposed mesentery was continuously superfused with bicarbonate buffer saline (pH 7.4, 37°C, 2 ml/min) and observed through an orthostatic microscope equipped with a video camera. Images were captured on videotape for playback analysis (final magnification of the video screen was x1300).

The mesentery was left to stabilize for a period of 30 min. When the agent to be tested was administered before the intravital microscopy experiment, images of two to three unbranched mesenteric venules (with diameters between 25 and 40 µm) were recorded for a period of 5 min per venule. When the agent was administered during the intravital microscopy experiment, a single venule was selected and images were recorded for 5 min periods at baseline (time 0) and at 15 min intervals over a 60 min period after administration.

The numbers of rolling, adherent, and emigrated leukocytes were determined off-line during playback analysis of videotaped images. Rolling leukocyte flux was assessed by counting the number of leukocytes passing a reference point in the vessel per minute. Leukocyte rolling velocity (Vwbc) was calculated by measuring the time required for a leukocyte to traverse a distance of 100 µm along the length of the venule and was expressed as micrometers per second. A leukocyte was considered to be adherent to the venular endothelium if it remained stationary for a period equal to or exceeding 30 s. Adherent cells were expressed as the number of white blood cells per 100 µm of venule. Leukocyte emigration induced by chronic treatments was evaluated as the total number of interstitial leukocytes per field. When the proinflammatory agent was administered acutely, leukocyte emigration was determined as the number of interstitial leukocytes in close proximity to the selected vessel (50 µm above and below the vessel). Systemic arterial blood pressure, venular diameters, and centerline red blood cell velocity were evaluated on-line, and venular blood flow and venular wall shear rate ({gamma}) were calculated as described previously (24) .

Gastrin concentration was measured by radioimmunoassay in plasma from the portal blood collected in heparine at the end of the experiment. In some cases, the area of the mesentery selected for the experiment was excised, fixed with paraformaldehyde (4% in PBS, pH 7.4) and stained with hematoxylin and eosin. The preparation was subsequently observed under a clear field microscope (x40), and the infiltrated leukocytes were counted (number per 0.5 cm2) and classified morphologically into PMN leukocytes, macrophages and lymphocytes by an observer who was unaware of the treatment previously given.

Experimental design
Effects of exogenous gastrointestinal hormones on leukocyte-endothelial cell interactions
The effects of gastrin (0.01–10 nM), pentagastrin (1 nM), CCK (1–100 nM), and somatostatin (1 µM) on leukocyte-endothelial cell interactions were evaluated using intravital microscopy. Each of these agents was added to the superfusion buffer after baseline measurements were taken, and their effects were evaluated over the following 60 min.

Some animals were pretreated before gastrin (1 nM) or CCK (10 nM) superfusion with an antagonist of the CCK-2 receptor (proglumide, 30 mg/kg, i.p.; or L-365,260, 1 mg/kg, i.v.) or with the CCK-1 receptor antagonist devazepide (1 mg/kg, i.v.).

To analyze whether gastrin was acting through the endogenous release of histamine, the H1 receptor antagonist diphenhydramine (2 µM) was cosuperfused with gastrin (1 nM). The involvement of mast cell activation was evaluated by administering the mast cell stabilizing agent cromolyn before initiating surgery (20 mg/kg, i.v.) and thereafter cosuperfusing 0.33 mg/ml of the drug with gastrin (1 nM). The effect of gastrin (1 nM) on mast cell activation was also analyzed by supplementing the superfusion buffer with 0.001% ruthenium red, which is captured by activated mast cells (25) . Superfusion with compound 48/80 at 1 µg/ml was used as a positive control of mast cell activation.

The selected doses of antagonists were taken from previous in vivo studies using either proglumide (26) , L-365,260 (27) , devazepide (27 28 29) , diphenhydramine (30) , or cromolyn (31) . The dose of proglumide used all along this study did not have any effect on the increase of leukocyte-endothelial interactions induced by a common proinflammatory agent like PAF (10–7M, data not shown).

Acute effects of endogenous gastrin on leukocyte-endothelial cell interactions
The endogenous release of gastrin was stimulated by two different procedures. In a first set of experiments, an intravenous infusion of gastrin releasing peptide (GRP, 300 pmol/kg/h) or saline was initiated after measurement of baselines and leukocyte-endothelial cell interactions were evaluated during the following 60 min. A second group of animals was treated with a maximal acid-inhibitory dose of the proton pump inhibitor omeprazole (40 mg/kg, p.o.), and leukocyte parameters were measured 5 h later, coinciding with the peaking of the gastrin plasma level (32) . When necessary, rats were pretreated with proglumide (30 mg/kg, i.p., –30 min).

Effects of an HP extract on leukocyte-endothelial cell interactions
The extract of HP was prepared from a strain CagA+/VacA+ (NCTC 11638). The bacteria were grown in Brucella broth culture (107 CFU/plate) supplemented with 5% FBS in a carbon dioxide incubator and maintained at 37°C for 5 days. The HP organisms were harvested in 1 ml of 0.15 M NaCl and centrifuged at 3,000 g for 25 min at 25°C as described previously (33 , 34) . The pellet was then resuspended in an equal volume of sterile distilled water, which was vortex-mixed for 45 s and centrifuged at 3,000 g. The resulting supernatant, containing water-extracted surface proteins, was finally filtered using a nylon mesh (0.2 µm) and stored at –20°C. The extracts were diluted with saline to normalize the total protein content to 100 µg/ml.

Rats received either the aqueous extract of HP or saline on days 1, 3, 5, and 7 (1 ml/rat, p.o.), and leukocyte-endothelial cell interactions were evaluated on day 14 (33) . Some animals were administered daily with the CCK-2 receptor antagonist proglumide (30 mg/kg/day, i.p., 14 days).

Detection of CCK-2 receptor mRNA by reverse transcriptase-polymerase chain reaction
CCK-2 receptor mRNA expression was analyzed in the mesenteric tissues and in peritoneal cell suspensions from control rats and in animals suffering an acute peritoneal inflammation after a zymosan injection (10 mg/rat in PBS, i.p.). In the latter case, peritoneal inflammatory cells were collected 4 h later by means of an abdominal lavage with 20 ml of ice-cold PBS medium containing 2 mM EDTA (35) . Rat gastric corpus was used as a positive control.

Total RNA from mesenteric and gastric tissues or from peritoneal cell suspensions was isolated with TriPure Isolation Reagent (Roche Diagnostics, Indianapolis, IN) or with RNeasy Mini Kit (Quiagen, Valencia, CA), respectively, and was treated with a DNA-free kit (Ambion, Austin, TX) to eliminate the possible traces of contaminating genomic DNA. Reverse transcription of this RNA was performed with SuperScript reverse transcriptase (RT; Invitrogen, Carlsbad, CA), using 0.5 µg oligo(dT16) and 40 U of RNase inhibitor (Roche Diagnostics, Indianapolis, IN). Minus-RT controls were included for each sample. For nested-polymerase chain reaction (PCR), reactions were produced in a LightCycler instrument (Roche Diagnostics) with the aid of a LightCycler-FastStart DNA Master SYBR Green I kit (Roche Diagnostics). The first round of PCR (annealing temperature: 57°C, 35 cycles) from cDNA was performed with specific primers for CCK-2 receptor (36) : 5'-CTTCATCCCGGGTGTGGTTATTGCG-3' (sense) and 5'-CCCCAGTGTGCTGATGGTGGTATAGC-3' (antisense), generating a product of 669 bp. After purification with Montage PCR (Millipore, Bedford, MA), PCR products were used as templates for the second round of PCR (annealing temperature: 60°C, 25 cycles) with internal primers designed in our laboratory: 5'-TGGCCTATGGACTCATCTCC-3' (sense) and 5'-AGCAGCCATCACTGTCTTCC-3' (antisense), which give rise to a product of 189 bp. Specificity of PCR was confirmed by melting curve analysis, agarose gel electrophoresis and sequenciation of the amplified product.

Immunohistochemical studies
Mesenteric windows were extracted, cleared of surrounding fat, whole-mounted on gelatin-coated slides, fixed for 10 min with paraformaldehyde (4% in PBS pH 7.4), washed with PBS, and immersed in methanol (–20°C) for 5 min. After antigen retrieval with alpha-chymotrypsin (Sigma Chemical CO) and blocking (10% goat serum, 1% BSA), specimens were incubated with a rabbit polyclonal anti–cholecystokinin-B receptor antibody (Ab; Abcam, Cambridge, MA, 1:100, 4°C, overnight). A goat anti-rabbit HRP conjugate (Southern Biotech, Birmingham, AL, 1:100) was used as secondary Ab and was incubated for 1 h at room temperature. Finally, tissues were incubated with 3,3'-diaminobenzidine (DAB) Enhanced Liquid substrate System for Immunohistochemistry (Sigma Chemical, St. Louis, MO). In some cases, mesenteric macrophages were identified by applying an analogous protocol to a second sample from the same animal but changing the primary Ab for a mouse monoclonal anti-macrophage + granulocyte Ab ([OX41], Abcam, 1:400) or a mouse monoclonal antibody (mAb) against an ED2-like antigen ([HIS36], eBioscience, San Diego, CA, 1:100).

All experimental protocols were performed according to the guidelines approved by the Ethical Committee for Experimental Research of the Faculty of Medicine of the University of Valencia.

Drugs
Gastrin, CCK, pentagastrin, somatostatin, GRP, omeprazole, proglumide, diphenhydramine, cromolyn, compound 48/80, and zymosan were all obtained from Sigma Chemical. Pentobarbital was from B. Braun Medical SA (Rubi, Barcelona, Spain). L-365,260 and devazepide were kindly donated by ML Laboratories PLC (Warrington, UK).

Statistical Analysis
All values are mean ± SEM. Data within groups were compared using an ANOVA (one-way ANOVA) followed by a Newman-Keuls post hoc test. The differences were considered significant when the P value was <0.05.

RESULTS

Acute effects of agonists and antagonists of CCK-gastrin receptors on leukocyte-endothelial cell interactions
Mesenteric venules exposed to gastrin superfusion experienced a concentration- and time-dependent increase in leukocyte-endothelial cell interactions. Gastrin increased the number of rolling leukocytes, while decreasing their rolling velocity. This effect was rapid in onset (15 min) and was observed with all the gastrin concentrations used (0.01-1 nM). This response was followed by significant increases in the number of adherent leukocytes and leukocytes emigrated into the interstitial tissue. These two effects were observed principally after superfusion with gastrin 0.1 and 1 nM (Fig. 1 ). Similar effects were induced by superfusion with pentagastrin 1 nM (rolling flux(60 min): 70±18, rolling velocity(60 min): 50±4; adhesion(60 min): 10±2; emigration(60 min): 2.3±0.3, P<0.05 vs. buffer and P<0.05 vs. basal values in all cases). Mesenteric superfusion with CCK also increased the interaction between leukocytes and venular endothelial cells (Fig. 2 ). The results included in Figs. 1 and 2 correspond to the maximal effects of these hormones (induced by gastrin 1 nM and CCK 10 nM) since similar responses were obtained by 10-fold increased concentrations (data not shown).


Figure 1
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  		Figure 1. Effects of exogenous gastrin on leukocyte-endothelial cell interactions in rat mesentery. Gastrin superfusion induced a concentration- and time-dependent increase in leukocyte rolling flux (A), adhesion (C), and emigration (D), with a parallel reduction in rolling velocity (B), in mesenteric postcapillary venules. Animals were divided into 4 groups: buffer, gastrin 0.01, 0.1, and 1 nM, and parameters were measured at 0, 15, 30, and 60 min. Results are mean ± SEM. *P< 0.05, **P < 0.01 vs. respective basal value (0 min).


Figure 2
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  		Figure 2. Effects of exogenous CCK on leukocyte-endothelial cell interactions in rat mesentery. CCK superfusion induced a concentration- and time-dependent increase in leukocyte rolling flux (A), adhesion (C), and emigration (D), with a parallel reduction in rolling velocity (B), in mesenteric postcapillary venules. Animals were divided into 3 groups: buffer, CCK 1 and 10 nM, and parameters were measured at 0, 15, 30, and 60 min. Results are mean ± SEM. **P < 0.01 vs. respective basal value (0 min).

Pretreatment with proglumide prevented both the increase in leukocyte rolling, adhesion, and emigration and the reduction in rolling velocity induced by gastrin (1 nM). The proinflammatory action of gastrin was also inhibited by pretreatment with another CCK-2 receptor antagonist, L-365,260. However, pretreatment with the CCK-1 antagonist devazepide failed to modify the effects of gastrin on leukocyte-endothelial cell interactions (Fig. 3 ). In a similar way, the proinflammatory effects of CCK (10 nM) were prevented by proglumide rather than devazepide (Fig. 4 ).


Figure 3
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  		Figure 3. Effects of CCK/gastrin receptor antagonists on gastrin-induced leukocyte/endothelium interactions. Rats were pretreated with two different CCK-2 receptor antagonists (proglumide 30 mg/kg, i.p.; or L-365,260 1 mg/kg, i.v.) or with a CCK-1 receptor antagonist (devazepide 1 mg/kg, i.v.). Rolling flux (A), rolling velocity (B), adhesion (C), and emigration (D) were evaluated in mesenteric postcapillary venules superfused with gastrin 1 nM for 60 min. Results are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. buffer; +P < 0.05, ++P < 0.01, +++P < 0.001 vs. gastrin 1 nM.


Figure 4
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  		Figure 4. Effects of CCK-gastrin receptor antagonists on CCK–induced leukocyte/endothelium interactions. Rats were pretreated with a CCK-2 receptor antagonist (proglumide 30 mg/kg, i.p.) or a CCK-1 receptor antagonist (devazepide 1 mg/kg, i.v.). Rolling flux (A), rolling velocity (B), adhesion (C), and emigration (D) were measured before (time 0) and 15, 30, and 60 min after the initiation of CCK superfusion. Results are mean ± SEM. +P < 0.05, ++P < 0.01, +++P < 0.001 vs. untreated group.

The proinflammatory effects induced by gastrin (1 nM) were unaltered by the addition of somatostatin 1 µM to the superfusion buffer (Table 1 ). This concentration of somatostatin did not affect per se any of the leukocyte parameters (rolling flux(60 min): 23±4, rolling velocity(60 min): 108±11; adhesion(60 min): 2.6±0.5; emigration(60 min): 0.8±0.2). Gastrin effects were not modified by either administration of an antagonist of the H1 receptor for histamine (diphenhydramine) or stabilization of mast cells with cromolyn (Table 1) . Furthermore, superfusion with gastrin did not induce ruthenium red uptake in mast cells.


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  		Table 1. Effects of somatostatin, an H1 receptor antagonist (diphenhydramine), or a mast cell stabilizing agent (cromolyn) on gastrin-induced leukocyte-endothelial cell interactions in rat mesenteric postcapillary venulesa

Basal hemodynamics were similar in all groups and none of the treatments produced significant changes in these parameters.

Acute effects of endogenous gastrin on leukocyte-endothelial cell interactions
GRP infusion significantly increased portal gastrinemia (36±5 vs. 23±2 pM in control animals, P<0.05) and leukocyte-endothelial cell interactions, which were reduced by pretreatment with proglumide (Fig. 5 ). Similarly, omeprazole pretreatment resulted in hypergastrinemia (68±8 vs. 20±1 pM in control animals, P<0.05) and augmented interactions between leukocytes and the venular endothelium, which were also prevented by proglumide (Fig. 5) . None of these treatments affected the hemodynamic parameters.


Figure 5
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  		Figure 5. Leukocyte-endothelial cell interactions induced by two drugs inducing acute hypergastrinemia and effects of a CCK-2 receptor antagonist. Rats were treated with gastrin releasing peptide (GRP, 300 pmol/kg/h, i.v. infusion) or omeprazole (40 mg/kg, p.o.) and their effects on leukocyte rolling flux (A), rolling velocity (B), adhesion (C), and emigration (D) were analyzed in mesenteric postcapillary venules 60 min after the initiation of GRP/saline i.v. infusion or 5 h after omeprazole/vehicle administration. Some animals were pretreated with proglumide (30 mg/kg, i.p.). *P < 0.05 and ***P < 0.001 vs. respective control; +P < 0.05, ++P < 0.01 and +++P < 0.001 vs. GRP + vehicle of proglumide group; #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. omeprazole + vehicle of proglumide group. (saline i.v.: n=7, GRP: n=10, proglumide+GRP: n=5, vehicle p.o.: n=14, omeprazole: n=13, proglumide+omeprazole: n=6).

HP extract causes mesenteric inflammation. Effect of a CCK-2 receptor antagonist
Rats chronically administered with an aqueous extract of a CagA+/VacA+ strain of HP showed an active inflammatory response in the mesenteric tissue as denoted by a higher number of rolling leukocytes moving at a slower rate, an increased leukocyte adhesion, and a significant increase in the number of leukocytes emigrated into the interstitium surrounding the selected venules (Fig. 6 ). Analysis of the hematoxylin and eosin-stained mesenteric tissue also revealed the presence of an inflammatory infiltrate in HP-treated animals (322±20 leukocytes per field in HP-treated rats vs. 44±2 leukocytes per field in control animals, P<0.001), consisting mainly of PMN leukocytes (79±1%), while macrophages and lymphocytes amounted to 15 ± 1 and 6 ± 1% of the total, respectively.


Figure 6
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  		Figure 6. Leukocyte-endothelial cell interactions induced by the HP extract and effect of a CCK-2 receptor antagonist. Rats were treated chronically (days 1, 3, 5, and 7) with the aqueous extract of HP or saline and their effects on leukocyte rolling flux (A), rolling velocity (B), adhesion (C), and emigration (D) were analyzed in mesenteric postcapillary venules on day 14. Animals were divided into 4 groups: saline + vehicle of proglumide (n=7), saline + proglumide (n=5), HP extract + vehicle of proglumide (n=5), and HP extract + proglumide (n=7). Results are mean ± SE. *P < 0.05 and ***P < 0.001 vs. saline + vehicle of proglumide group; +P < 0.05 and ++P < 0.01 vs. HP extract + vehicle of proglumide group.

Chronic administration of the aqueous extract of HP also caused hypergastrinemia (39±7 vs. 19±1 pM gastrin in control rats, P<0.05), and daily treatment of these animals with the CCK-2 receptor antagonist proglumide significantly reduced the number of rolling, adherent and emigrated leukocytes (Fig. 6) . Hemody namic parameters were similar in all groups. Hematoxylin and eosin staining revealed that rats receiving proglumide also presented an inflammatory infiltrate but with fewer leukocytes (244±12 vs. 322±20 leukocytes per field, P<0.01) and differing proportions of each leukocyte type. In contrast to what was observed in HP-treated rats (see previous paragraph), the infiltrate in rats receiving proglumide contained similar numbers of PMN leukocytes and macrophages (44±3 and 50±4%, respectively; Fig. 7 ).


Figure 7
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  		Figure 7. Leukocyte infiltration in the mesentery of rats treated with the HP extract and effect of a CCK-2 receptor antagonist. Rats received saline + vehicle of proglumide (A), HP extract + vehicle of proglumide (B), saline + proglumide (C), or HP extract + proglumide (D). Chronic administration (days 1, 3, 5, and 7) of the aqueous extract of Helicobacter pylori induces the formation of an inflammatory infiltrate in the rat mesentery mainly composed of PMN leukocytes (B). Daily treatment with proglumide reduces the number of extravasated leukocytes and increases relative amount of macrophages in the inflammatory infiltrate (D). Tissues are stained with hematoxylin and eosin. Arrows denote mesenteric macrophages. Bar = 50 µm.

Analysis of the expression of the CCK-2 receptor
CCK-2 receptor mRNA, analyzed by nested RT-PCR, was detected in both mesenteric tissues of control rats and peritoneal cell suspensions of zymosan-treated rats (Fig. 8 ).


Figure 8
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  		Figure 8. Expression of CCK-2 receptor mRNA in rat mesentery. CCK-2 receptor mRNA is present in mesenteric tissues of control rats and peritoneal cell suspensions obtained from zymosan-treated rats as analyzed by nested RT-PCR. Lane 1, 100 bp MW marker; lane 2, cDNA of gastric corpus; lane 3, -RT control; lane 4, cDNA of mesenteric tissues; lane 5, -RT control; lane 6, cDNA of peritoneal cells; lane 7, -RT control; lane 8, water. Specificity of reaction was assessed through agarose gel electrophoresis (this figure), melting curve analysis and sequenciation of the amplified product.

Immunohistochemical studies revealed the presence of CCK-2 receptor in two cellular types of mesenteric tissue, which were later identified in the hematoxylin and eosin staining as PMN leukocytes and macrophages. The identity of macrophages was further confirmed by comparing the morphology of non-PMN CCK-2 receptor positive cells with cells stained with two different macrophage membrane markers in parallel immunohistochemical studies (Fig. 9 ). Endothelial cells tested negative for the CCK-2 receptor immunostaining.


Figure 9
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  		Figure 9. Immunostaining for CCK-2 receptor in rat mesentery. Positive staining is evident in macrophages (A) and PMN leukocytes (B). No staining was observed in the absence of primary Ab (not depicted). Insets in A correspond to immunostaining for 2 different macrophage markers: A) monoclonal anti-macrophage + granulocyte Ab [OX41], Abcam; B) mAb against an ED2-like antigen [HIS36], eBioscience).

DISCUSSION

The present study demonstrates for the first time that gastrin has a proinflammatory action in vivo and suggests that this peptide may directly contribute to the inflammatory response elicited by HP.

We have observed by intravital microscopy that gastrin enhances the interactions between the flowing leukocytes and the venular endothelium in rat mesentery. Gastrin increases the number of rolling leukocytes, while it reduces their rolling velocity and enhances leukocyte adhesion and emigration into the interstitium. Since these are the initial steps in the formation of inflammatory foci, our results indicate that gastrin induces a proinflammatory activity. As said before, peptides of the gastrin family mediate their effects on target tissues by activating two different receptors named CCK-1 and CCK-2. Our results suggest that the proinflammatory activity of gastrin is a CCK-2 receptor-mediated process since it can be reversed by its specific antagonists proglumide and L-365,260 but not by the CCK-1 antagonist devazepide. Significant increases in leukocyte rolling, adhesion, and emigration were also observed with pentagastrin and CCK superfusion and when the endogenous release of gastrin was stimulated by the neuropeptide GRP or the potent antisecretory drug omeprazole. In all cases, these events were partially reversed by pretreatment with proglumide.

CCK-2 receptor mRNA was detected in rat mesenteric tissue extracts and, through immunohistochemistry studies, those receptors were localized in mesenteric macrophages and PMN leukocytes. Likewise, CCK-2 receptor mRNA was also present in extracts from leukocyte suspensions obtained by peritoneal lavage of zymosan-treated rats, thus confirming the location of the receptor in rat inflammatory cells. These results are similar to those previously reported for humans (3 4 5 6 7 8) . CCK-2 receptors are G protein-coupled receptors that, on binding to their peptide agonists, activate proteins of the G{alpha}q or G{alpha}12,13 subfamilies (37 , 38) . The signaling pathways initiated by these proteins share several elements with those that lead to leukocyte activation after chemokine binding to their G protein-coupled receptors or after ß2-integrin engagement (39 40 41 42) . Although it is beyond the scope of the present study, we suspect that some of these elements account for the proinflammatory activity of gastrin that we observed in vivo and/or the modulation of leukocyte adhesion and emigration previously demonstrated in vitro (11 , 43) .

In the HP-infected mucosa both elements, significant gastrin levels (16 17 18) and infiltrated leukocytes can be found, and, considering the effects observed with gastrin, a modulatory effect of the peptide on the local leukocytes function should be expected. Natural colonization by HP is observed exclusively in humans and primates, and different strategies have been used to get valid murine and rat models of infection. We have administered an extract obtained from bacterial suspensions containing many of the pathogenic components of the germ (44) . Similar HP extracts have previously been shown to induce an acute inflammatory response in the digestive tract (45 46 47) , which is generally attributed to a direct effect of bacterial components on leukocytes (34 , 45 , 48 49 50) . In our experimental design, the extract was administered for 1 wk, and the experiments were performed 7 days later, when increased gastrin plasma levels were detectable (33 , this study). At this stage, despite the suspension of administration of the extract during a relatively long period, there was still an active inflammatory process in the gastrointestinal tract. We observed increased leukocyte rolling, adhesion, and emigration in the mesenteric venules of rats receiving the HP extract, which coincides with the high levels of proinflammatory cytokines seen in plasma from animals following the same protocol (33) . It is important to note that daily treatment with the antagonist proglumide significantly reduced the leukocyte-endothelial cell interactions induced by the HP extract and the number of interstitial leukocytes. On this basis, it can be speculated that gastrin, released in response to HP-induced inflammation, aids the recruitment of leukocytes by activating local macrophages or granulocytes, which in turn would contribute to the persistence of the inflammatory process. In fact, when this activity is impeded by proglumide, the rat mesentery presents an altered pattern of infiltration, with more macrophages and less PMN leukocytes, suggestive of a receding inflammatory process. It was previously observed in HP-infected patients a direct relationship between gastrinemia and the number of mucosal PMN (16) and mononuclear (16 , 17) leukocytes. At the time, and based on the observed stimulatory effect of proinflammatory cytokines and activated monocytes on gastrin release from cultured antral G cells (19 20 21) , this correlation was explained as hypergastrinemia being the consequence of the inflammatory process. Our results complement this idea by suggesting that gastrin released in response to local inflammation exerts a positive feedback on the inflammatory process.

Gastrin stimulates gastric acid secretion not only by direct activation of CCK-2 receptors on parietal cells but also through the release of histamine from enterochromaffin-like cells within the gastric mucosa (1) . Histamine is a mediator of acute inflammatory reactions, helping to recruit rolling leukocytes and increasing albumin leakage in postcapillary venules via H1 receptor activation (51) . The main sources of histamine in our experimental preparation are mastocytes, and there is some evidence suggesting that activation of the CCK-2 receptor induces mast cell degranulation in the intestine (52) . Our experiments, however, indicate that histamine release is not involved in the proinflammatory action of gastrin since it was unaffected by stabilization of mastocytes with cromolyn or blockade of H1 receptors with diphenhydramine.

The acid secretory effect of gastrin is physiologically counteracted by somatostatin, the dual action of which directly inhibits parietal cells and reduces the release of gastrin from G cells. HP decreases somatostatin synthesis (15 , 53) , which could partly explain the gastrin hypersecretion, and somatostatin displays anti-inflammatory properties (54 55 56 57) . Thus, gastrin proinflammatory activity may be limited by the physiological release of this peptide or enhanced when its secretion is reduced by HP infection. However, in the present study, somatostatin did not alter in any way the basal leukocyte-endothelial cell interactions nor did it reduce the proinflammatory action of gastrin, which rules out a regulatory role for somatostatin in this process.

In summary, our results reveal that gastrin has a proinflammatory effect and suggest that local endocrine G cells exposed to HP-induced inflammation secrete more gastrin and contribute to the progression of the focus. Further research is required to define the relevance of this effect of gastrin in other pathophysiological contexts.

ACKNOWLEDGMENTS

The present study was awarded the 2002 Prize of Salvat Foundation from Salvat Laboratories and has been supported by grants PI020461 and C03/02 from Ministerio de Sanidad y Consumo, SAF2004–06211 and SAF2005–01366 from Ministerio de Educación y Cultura and 05/043 from Generalitat Valenciana. The authors thank Dra. M. A. Ferrús (Grupo de Investigación sobre Helicobacter, Dpto. de Biotecnología, Universidad Politécnica de Valencia) for preparing the HP extract.

Received for publication January 31, 2006. Accepted for publication June 2, 2006.

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