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* Departamento de Farmacología and
Unidad Mixta CNIC-UVEG, Facultad de Medicina, Universidad de Valencia, Valencia, Spain; and
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
SPECIFIC AIMS
Infection by Helicobacter pylori (HP), the main cause of nonautoimmune chronic gastritis, increases gastrin secretion, and a correlation between gastrinemia and the severity of gastritis has been identified. Gastrin receptors have been observed in several leukocytes, and gastrin and cholecystokinin (CCK) have been shown to induce changes in leukocyte functions such as chemotaxis, adherence, or phagocitosis in vitro. However, it has not been analyzed whether these cellular effects of gastrin have any consequence on the functioning of the immune system in vivo. If this was the case, 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.
PRINCIPAL FINDINGS
1. Gastrin and related peptides induce leukocyte-endothelial cell interactions
Gastrin superfusion induced a concentration- and time-dependent increase in leukocyte-endothelial cell interactions in the mesenteric venules as evaluated by intravital microscopy in anesthetized rats. Gastrin increased the number of rolling leukocytes, while decreased their rolling velocity. This effect was rapid in onset (15 min) and was observed with all the gastrin concentrations used (0.01-1nM). This response was followed by significant increases in the number of adherent leukocytes and leukocytes emigrated into the interstitial tissue. Similar effects were induced by superfusion with pentagastrin (1nM) or CCK (1-10nM).
2. Effects of gastrin and CCK on leukocyte-endothelial cell interactions are mediated through CCK-2 receptors
Pretreatment with proglumide (30 mg/kg, i.p.) prevented both the increase in leukocyte rolling, adhesion, and emigration and the reduction in rolling velocity induced by gastrin (1nM). These effects of gastrin were also inhibited by pretreatment with another CCK-2 receptor antagonist L-365,260 (1 mg/kg, i.v.) but unaffected by pretreatment with the CCK-1 antagonist devazepide (1 mg/kg, i.v.). In a similar way, the increase in leukocyte-endothelial cell interactions induced by CCK (10nM) was prevented by proglumide and not by devazepide.
Endogenous release of gastrin was stimulated by an intravenous infusion of gastrin-releasing peptide (300 pmol/kg/h) or by a single maximal dose of the antisecretory drug omeprazole (40 mg/kg, p.o.), and both treatments induced significant increases in leukocyte-endothelial cell interactions, which were also prevented by pretreatment with proglumide.
3. Analysis of the expression of the CCK-2 receptor
CCK-2 receptor mRNA, analyzed by nested reverse transcriptase-polymerase chain reaction, was detected in both mesenteric tissues of control rats and peritoneal cell suspensions of zymosan-treated rats. Immunohistochemical studies revealed the presence of CCK-2 receptor in two cellular types of mesenteric tissue, which were identified as polymorphonuclear leukocytes and macrophages. Endothelial cells tested negative for the CCK-2 receptor immunostaining.
4. Effects of gastrin on leukocyte-endothelial cell interactions are not mediated by histamine nor modulated by somatostatin
The increase in leukocyte-endothelial cell interactions induced by gastrin (1nM) was not modified by either an antagonist of the H1 receptor for histamine diphenhydramine (2 µM cosuperfused) or stabilization of mast cells with cromolyn (20 mg/kg, i.v. plus cosuperfusion with 0.33 mg/ml). The effects of gastrin (1nM) were also unaltered by the addition of somatostatin (1nM) to the superfusion buffer.
5. HP extract causes mesenteric inflammation. Effects of a CCK-2 receptor antagonist
The extract of HP was prepared from a strain CagA+/VacA+ (NCTC 11638) and was administered on days 1, 3, 5, and 7 (1 ml/rat, p.o.). Leukocyte-endothelial cell interactions were evaluated on day 14. Rats receiving the HP extract 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. 1
). Analysis of the eosin and hematoxylin-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 polymorphonuclear leukocytes (79±1%), whereas macrophages and lymphocytes amounted to 15 ± 1 and 6 ± 1% of the total, respectively.
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. 1)
. Eosin and hematoxylin 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 polymorphonuclears and macrophages (44±3 and 50±4%, respectively).
CONCLUSIONS AND SIGNIFICANCE
The present study demonstrates for the first time that gastrin has a proinflammatory action in vivo and points to a role for gastrin hypersecretion in the inflammatory reaction caused by HP.
Gastrin enhances the interactions between the flowing leukocytes and the venular endothelium in rat mesentery, increasing the number of rolling, adherent, and emigrated leukocytes. These are the initial steps conducting to the formation of inflammatory foci and, thus, our results indicate that gastrin possess a proinflammatory activity. Similar responses were obtained with pentagastrin and CCK and when the endogenous release of gastrin was stimulated by the neuropeptide GRP or the potent antisecretory drug omeprazole. Gastrin and CCK mediate their effects on target tissues by activating the G protein-coupled CCK-1 and CCK-2 receptors. Their proinflammatory activity seems to be 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. The CCK-2 receptor mRNA was detectable in rat mesenteric tissue extracts, and immunohistochemistry studies localized those receptors in mesenteric macrophages and polymorphonuclear leukocytes.
Gastrin partly stimulates gastric acid secretion through the release of histamine from enterochromaffin-like cells. Histamine is a mediator of acute inflammatory reactions, and there is some evidence suggesting that activation of the CCK-2 receptor may degranulate mast cells, an important source of histamine in the mesentery. However, our experiments 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, and some anti-inflammatory properties have been attributed to this peptide. Nevertheless, somatostatin did not modify the effects of gastrin, which rules out a regulatory role for somatostatin in its proinflammatory action.
In the HP-infected mucosa, significant gastrin levels and infiltrated leukocytes can be found and, considering the effects observed with gastrin, a modulatory effect of the peptide on local leukocytes function should be expected. We have administered an extract obtained from bacterial suspensions containing many of the pathogenic components of the germ for 1 wk and the experiments were performed 7 days later, when increased gastrin plasma levels were detectable. At this stage, 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 (Fig. 1)
, while high levels of proinflammatory cytokines in plasma were seen in previous studies following the same protocol. 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 polymorphonuclear 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 polymorphonuclear and mononuclear 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, 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 (Fig. 2
).
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.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5696fje
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  A. Alvarez, M. S. Ibiza, M. M. Andrade, A. Blas-Garcia, and S. Calatayud Gastric Antisecretory Drugs Induce Leukocyte-Endothelial Cell Interactions through Gastrin Release and Activation of CCK-2 Receptors J. Pharmacol. Exp. Ther., October 1, 2007; 323(1): 406 - 413. [Abstract] [Full Text] [PDF]
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