Brief Reports
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World J Gastroenterol. Mar 14, 2005; 11(10): 1549-1553
Published online Mar 14, 2005. doi: 10.3748/wjg.v11.i10.1549
Helicobacter pylori strain-specific modulation of gastric inflammation in Mongolian gerbils
Ken Ohnita, Hajime Isomoto, Shoji Honda, Akihiro Wada, Chun-Yang Wen, Yoshito Nishi, Yohei Mizuta, Toshiya Hirayama, Shigeru Kohno
Ken Ohnita, Hajime Isomoto, Yoshito Nishi, Yohei Mizuta, Shigeru Kohno, Second Department of Internal Medicine, Nagasaki University School of Medicine, Sakamoto 1-7-1, Nagasaki, Japan
Shoji Honda, Second Department of Internal Medicine, Oita Medical University, 1-1 Hasama, Oita 987-5593, Japan
Akihiro Wada, Toshiya Hirayama, Department of Bacteriology, Institute of Tropical Medicine, Nagasaki University School of Medicine, Sakamoto 12-4, Nagasaki, Japan
Chun-Yang Wen, Department of Molecular Pathology, Atomic Bomb Disease Institute, Nagasaki University School of Medicine, Sakamoto 12-4, Nagasaki, Japan
Author contributions: All authors contributed equally to the work.
Correspondence to: Dr. Hajime Isomoto, Second Department of Internal Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki, Japan. hajimei2002@yahoo.co.jp
Telephone: +81-95-849-7567 Fax: +81-95-849-7568
Received: September 2, 2004
Revised: September 3, 2004
Accepted: September 15, 2004
Published online: March 14, 2005

Abstract

AIM: The cag pathogenicity island (PAI) is one of potential virulence determinants of Helicobacter pylori. The Mongolian gerbil is a suitable experimental animal for the screening of virulence factors of H pylori.

METHODS: Five-week-old Mongolian gerbils were inoculated with a standard H pylori strain (ATCC 43504) possessing the cag PAI or a clinical isolate lacking the genes’ cluster (OHPC-0002). The animals were killed at 2, 4, 8, 24 and 48 wk after inoculation (n = 5 each), and macroscopic and histopathological findings in the stomachs were compared.

RESULTS: In gerbils infected with ATCC 43504, a more severe degree of infiltration of polynuclear and mononuclear cells and lymphoid follicles was observed from 4 wk after inoculation compared to gerbils infected with OHPC-0002 especially in the antrum and transitional zone from the fundic to pyloric gland area. In addition, glandular atrophy, intestinal metaplasia, gastric ulcer and hyperplastic polyps were noted in gerbils infected with ATCC 43504, whereas only mild gastric erosions occurred in those infected with OHPC-0002.

CONCLUSION: Our results indicate that the cag PAI could be directly involved in gastric immune and inflammatory responses in the Mongolian gerbils, leading to a more advanced gastric disease.

Key Words: Helicobacter pylori, Mongolian gerbil, cag pathogenicity island


Citation: Ohnita K, Isomoto H, Honda S, Wada A, Wen CY, Nishi Y, Mizuta Y, Hirayama T, Kohno S. Helicobacter pylori strain-specific modulation of gastric inflammation in Mongolian gerbils. World J Gastroenterol 2005; 11(10): 1549-1553
INTRODUCTION

Helicobacter pylori (H pylori) is a Gram-negative, spiral-shaped, microaerophilic bacterium that colonizes the human gastric mucosa[1-3]. The prevalence of H pylori infection is still high in Asian countries including Japan, and this organism is now recognized as a major cause of chronic active gastritis[1-4], peptic ulcers[2-6] and gastric malignancies[4,5,7,8]. However, the clinical manifestation of H pylori infection varies widely in infected individuals and most persons are asymptomatic all their life[9]. The pathogenic mechanism of this pleomorphism is not completely understood, but might be explained, in part, by strain diversity[10-13].

Several factors have been proposed as possible virulence determinants[14-18]. Among them, the cag pathogenicity island (cag PAI), a 40-kb cluster of -30 genes of supposedly extraneous origin[19], is reportedly associated with advanced gastroduodenal diseases including gastric cancer[11-14].

In an effort to develop a model that would parallel as close as possible the course of infection as it occurs in humans, Hirayama et al[20] established an experimental model of inbred Mongolian gerbils with persistent H pylori infection. Infection of Mongolian gerbils with H pylori results in chronic active gastritis, gastric ulcer, intestinal metaplasia and gastric cancer[21-24]. The Mongolian gerbil model may be valuable not only in elucidating H pylori-related neoplasia but also in evaluating virulence factors[11,12]. We sought to investigate the chronological and spatial changes of gastric inflammation after inoculation of H pylori strains possessing or lacking the cag PAI.

MATERIALS AND METHODS
Animals

Five-week-old male specific pathogen-free Mongolian gerbils (MGS/Sea, male) weighting 27-43 g (Seac Yoshitomi, Fukuoka, Japan) were used. They were housed four per cage in stainless-steel cages on hardwood chip bedding in an air-conditioned room (12/12 h light/dark cycle) at 24 °C. They were fasted for 24 h prior to H pylori inoculation, and then fed with chow (CE-2; Clea Japan Co., Tokyo, Japan) and distilled water ad libitum 12 h after inoculation. Experiments were performed according to the guidelines of Ethics Committee for Animal Experiments at Nagasaki University.

Bacteria

We used the ATCC 43504 standard strain of H pylori known to possess cag PAI and the cag PAI-totally deleted strain OHPC-0002[25]. The H pylori strains were cultivated on Muller Hinton II agar plate with 7% horse blood at 37 °C for 3 d under microaerophilic conditions[25]. Bacteria harvested from the plates were incubated in 200 mL of Brucella broth (DIFCO Laboratories, Detroit, MI) with 10% horse serum at 37 °C for 24 h with vigorous shaking under controlled microaerophilic atmosphere[23,25]. The inoculum size was adjusted with sterile saline to produce the optical density of McFarland 4 at 540 nm[23].

Experimental design

A total of 75 Mongolian gerbils were randomly assigned to three experimental groups (25 each group). The animals were challenged with the vehicle (group A), 109 colony-forming units (CFU) ATCC 43504 (group B) or OHPC-0002 (group C) in 1 mL of Brucella broth with 10% horse serum by intragastric gavage. Five gerbils in each experimental group were sacrificed under anesthesia at 2, 4, 8, 24 and 48 wk after inoculation. The stomachs were quickly removed, opened along the greater curvature, divided into two, and used for histopathological and culture examinations.

Histopathological examination

Half the stomach was fixed in 10% neutral buffered formalin and embedded in paraffin. Four µm thick sections were stained with hematoxylin and eosin. According to the Sydney system, each histological parameter of activity (neutrophils), chronic inflammation (mononuclear cells), glandular atrophy and intestinal metaplasia was graded into none, mild, moderate or severe[26]. Intestinal metaplasia was defined by the presence of goblet cells in glandular mucosa with Alcian blue (pH 2.5)/periodic acid-Schiff staining[27]. The biopsy specimens were examined blindly without knowledge of the results of the experimental group.

Culture study

The remaining half of the stomach was minced in 2 mL of Brucella broth, placed on the blood agar and incubated for 4 d at 37 °C under the microaerophilic conditions[23].

RESULTS
H pylori colonization

H pylori was recovered from almost all gerbils of groups B and C from 4 wk after inoculation throughout the whole observation period. The bacterial densities assessed by Giemsa staining did not differ significantly between groups B and C.

Macroscopic findings

There were no visible changes in the stomach of Mongolian gerbils of group A throughout the whole observation period. Until 4 wk after inoculation, no gloss appearance was noted in the stomach of both groups B and C. At 8 wk, erosions with bleeding appeared in the antrum and transitional zone extending from fundic to pyloric mucosa in all the animals of group B. In one gerbil of the group B, the stomach contained an ulcer in the vicinity of the transitional zone at 8 wk post-inoculation. The erosive lesions were noted in the transitional mucosa of 3 and 4 animals of group B at 24 and 48 wk respectively. Sessile polyps with occasional apical erosions, which were classified histopathologically as hyperplastic polyps, were seen in the antrum of one Mongolian gerbil of group B. On the other hand, there were no visible macroscopic findings in the stomach of Mongolian gerbils of group C until 24 wk after inoculation. Mild erosions were seen in the antrum of one gerbil and in the transitional zone of 2 gerbils of group C at 48 wk.

Histopathological findings

No histopathological changes were noted in the stomach of Mongolian gerbils of group A throughout the whole observation period. Table 1 compares the histopathological grades of gastritis noted in the mucosa of gerbils of groups B and C based on the Sydney classification. At 2 wk after inoculation, mild mucosal infiltration of polynuclear and mononuclear cells appeared in the antrum of one gerbil of group B. At 4 wk, moderate to severe chronic active gastritis consisting of mononuclear and polynuclear cells was noted in the lamina propria of the antrum and transitional zone of group B. The histopathological changes in group B reached peak levels between 8 to 24 wk (Figure 1A). Lymphoid follicles were especially conspicuous in the submucosa but they were also found in the deep portion of the mucosa of almost all animals of group B from 4 wk after inoculation. In the fundic mucosa of group B, mild mononuclear and polynuclear cell infiltration appeared at 4 wk. Mild to moderate infiltration of neutrophils and mononuclear cells was noted in the corpus of some gerbils of group B up to 48 wk, but the grades of gastritis were less severe than those in the antrum and the transitional zone. Mild atrophic gastritis appeared in the antrum and transitional zone of Mongolian gerbils of group B at 4 wk after inoculation, but seen in the corpus of only one gerbil at 24 wk. Intestinal metaplasia of incomplete type was found in the antrum of one gerbil of group B.

Table 1 Chronological and spatial changes of various parameters of histopathological gastritis in Mongolian gerbils infected with two species of H pylori.
Post-inoculation interval (wk)
Antrum
Transitional zone
Corpus
248244824824482482448
ATCC 43504-infected mongolian gerbils
Activity
None400015000153234
Mild122240201202220
Moderate032300224200101
Severe001000130000000
Chronic inflammation
None400005000052022
Mild100000200103313
Moderate021220331200220
Severe034330024200000
Glandular atrophy
None522225422155545
Mild033330133400010
Moderate000000000000000
Severe000000000000000
Intestinal metaplasia
None555545555555555
Mild000010000000000
Moderate000000000000000
Severe000000000000000
ohpc-0002-infected mongolian gerbils
Activity
None554535555355554
Mild001020000200001
Moderate000000000000000
Severe000000000000000
Chronic inflammation
None554215554355554
Mild001320001000001
Moderate000020000200000
Severe000000000000000
Glandular atrophy
None554345554355545
Mild001210001200010
Moderate000000000000000
Severe000000000000000
Intestinal metaplasia
None555555555555555
Mild000000000000000
Moderate000000000000000
Severe000000000000000
Figure 1
Figure 1 A: Severe chronic active gastritis was seen in the antrum of Mongolian gerbils infected with ATCC 43504 at 24 wk after inoculation (magnification, 40-fold); B: Mild chronic gastritis was noted in the antrum of Mongolian gerbils infected with OHPC-0002 at 24 wk after inoculation (magnification, 40-fold).

On the other hand, some Mongolian gerbils of group C showed mild mononuclear and polynuclear cell infiltration in the antral mucosa at 8 wk after inoculation (Figure 1B). Furthermore, mild to moderate chronic active gastritis was noted in the antrum and transitional zone at 48 wk in some animals. Glandular atrophy appeared in the antral and transitional mucosa at 8 wk but was exclusively mild. No intestinal metaplasia was present anywhere in the animals of group C.

DISCUSSION

Various animals have been used for studying experimental H pylori infection including monkeys, dogs, piglets, domestic cats, rats and nude mice[28-31]. These animal models had disadvantages such as low infection rates, instability, immunodeficiency and high costs[11,32]. Infection of conventional mice with H pylori strain SS1 overcame many of the above problems, but gastritis in mice induced by this infection is less intense than in humans[33]. In 1996, Hirayama et al[20] reported the development of a successful Mongolian gerbil model with persistent H pylori infection. Several investigators confirmed successively the progression from chronic active gastritis to atrophic gastritis and then to intestinal metaplasia and the occurrence of gastric ulcer in the Mongolian gerbil model[22,23]. In addition, long-term H pylori infection by itself resulted even in the development of gastric adenocarcinoma[24]. Thus, the gerbil seems to be the best experimental model for H pylori infection, and we employed the Mongolian gerbil to unravel the impact of cag PAI status, an important virulence determinant, on gastric pathogenesis in vivo.

In Mongolian gerbils infected with ATCC 43504 with total cag PAI, mild infiltration of neutrophils and mononuclear cells appeared in the antrum at 2 wk after inoculation. From 4 wk, moderate to severe chronic active gastritis, often accompanied by lymphoid follicle formation, was noted predominantly in the antrum and transitional zone. In the fundic mucosa, the severity of gastritis worsened to moderate from 8 wk. Such chronological changes of histological gastritis and spatial shift from antral to corporeal mucosa observed in gerbils infected with ATCC 43504 were consistent with previous results in Mongolian gerbils inoculated with other standard or clinical strains possessing intact cag PAI[11,12,22,23].

The major finding of the present study is that the strain possessing the cag PAI induced more severe degree of gastritis in Mongolian gerbils than clinical strain lacking the genes’ cluster[25]. Because there were no differences in the susceptibility and bacterial density between the two strains, ATCC 43504 and OHPC-0002, the deletion did not change the ability of bacterial colonization or proliferation but affected the inflammatory reactions in the stomach of Mongolian gerbils. Akanuma et al[12] cited similar results; a genetically handled mutant lacking total cag PAI or knockout strain of cagE, one of the functional genes in cag PAI, ameliorated histopathological gastritis in this animal. In addition, glandular atrophy, incomplete intestinal metaplasia, gastric ulcer and hyperplastic polyps were noted in gerbils infected with the cag PAI-positive strains, whereas only mild gastric erosions concomitant with slight infiltration of inflammatory cells occurred in animals infected with the cag PAI-negative strains. These findings indicate that intact cag PAI could be directly involved in the host immune and inflammatory processes and cause more advanced gastric diseases.

Deletions of the cag PAI and several cag insertion mutations reportedly block the induction of proinflammatory cytokine, interleukin-8, in gastric epithelial cell lines[19,34]. The expression of interleukin-8 gene is up-regulated by nuclear factor kappa B (NF-κB), which is involved in various inflammatory conditions such as inflammatory bowel diseases, rheumatoid arthritis and H pylori-associated gastritis through the regulation of a plethora of genes that encode bioactive molecules that mediate various immune and inflammatory responses[35-37]. In fact, accumulating evidence indicates that intact cag PAI is a prerequisite for the activation of NF-κB[34]. Considered together, these results suggest that the cag PAI-mediated activation of NF-κB plays an important role in the pathogenesis of gastritis in Mongolian gerbils.

The VacA protein, which causes vacuolation in cultured cells[17,18], is responsible for the virulence of H pylori and ulcer formation in the stomach and duodenum[10,38]. However, since the VacA-positive strains often have intact cag PAI, the involvement of VacA in inducing gastritis is still controversial[11,39]. In this regard, the VacA-disrupted H pylori strain could cause inflammatory changes in the stomach of Mongolian gerbils to a degree similar to those seen in Mongolian gerbils infected with the wild strain[11], indicating that VacA is not crucial for gastric inflammation. Several other candidate genes outside the cag PAI[15,16] should be validated in this reliable rodent model for the screening of determinants of virulence of H pylori.

Footnotes

Edited by Li WZ Language Editor Elsevier HK

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