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. 1998 Apr;66(4):1349–1355. doi: 10.1128/iai.66.4.1349-1355.1998

Immune Responses of Specific-Pathogen-Free Mice to Chronic Helicobacter pylori (Strain SS1) Infection

Richard L Ferrero 1,*, Jean-Michel Thiberge 1, Michel Huerre 2, Agnès Labigne 1
PMCID: PMC108059  PMID: 9529052

Abstract

A model permitting the establishment of persistent Helicobacter pylori infection in mice was recently described. To evaluate murine immune responses to H. pylori infection, specific-pathogen-free Swiss mice (n = 50) were intragastrically inoculated with 1.2 × 107 CFU of a mouse-adapted H. pylori isolate (strain SS1). Control animals (n = 10) received sterile broth medium alone. Animals were sacrificed at various times, from 3 days to 16 weeks postinoculation (p.i.). Quantitative culture of gastric tissue samples from inoculated mice demonstrated bacterial loads of 4.0 × 104 to 8 × 106 CFU per g of tissue in the animals. Infected mice had H. pylori-specific immunoglobulin M (IgM) and IgG antibodies in serum (at day 3 p.i.) and IgG and IgA antibodies in their gastric contents (weeks 4 and 16 p.i.) and saliva (week 16 p.i.). Mucosal IgM antibodies were not detected. Histological examination of the gastric mucosae from control and infected mice revealed mild chronic gastritis, characterized by the presence of polymorphoneutrophil cell infiltrates and submucosal lymphoid aggregates, in infected animals at 16 weeks p.i. Differences in the quantities of IgG1 and IgG2a subclass antibodies detected in the sera of mouse strains (Swiss, BALB/c, and C57BL/6) infected by H. pylori suggested that host factors influence the immune responses induced against this bacterium in the host. In conclusion, immune responses to H. pylori infection in mice, like those in chronically infected humans, appear to be ineffective in resolving the infection.

The presence of Helicobacter pylori bacteria in human gastric mucosae induces marked immune responses in the host (for a review, see reference 10). Volunteer ingestion experiments and case reports have shown that individuals develop severe polymorphonuclear leukocyte inflammation of the stomach mucosa soon after infection by H. pylori (23, 29). In addition, acutely infected individuals were reported to have anti-H. pylori immunoglobulin A (IgA) and IgM class antibodies in their gastric juice and/or sera within several weeks after having been infected (26, 27, 33). Though there has been some evidence of spontaneous eradication of H. pylori by the host (2, 26), most untreated individuals remain infected with the organism. In such cases, subjects develop a chronic gastritis which is characterized by the formation of gastric lymphoid tissue (10).

Various animal models have been developed for study of H. pylori pathogenesis, and, until recently, those using large animal hosts such as gnotobiotic piglets, nonhuman primates, and cats have been the most successful at reproducing the pathology associated with human infection (for a review, see reference 14). Nevertheless, such models are relatively cumbersome and have a restricted applicability because of difficulties in handling large numbers of infected animals for significant periods and because of the limited availability of immunological reagents for these host species.

In 1991, Karita and colleagues (18) established transient H. pylori infections in immunodeficient BALB/c animals, thus demonstrating for the first time that it was possible to colonize a small laboratory animal with H. pylori. More recently, there have been reports of the colonization of immunocompetent mice with mouse-adapted H. pylori isolates (19, 22, 24). By screening various H. pylori clinical isolates for their capacity to colonize mice, Lee and colleagues (21) identified one H. pylori strain (named SS1, or the Sydney strain) that, after adaptation to mice, was able to colonize mouse gastric mucosae in high numbers and for long periods (≤8 months).

Data on host immune responses to H. pylori in humans have, for the most part, arisen from investigations of chronically infected individuals (2, 5, 6, 31), while studies with animal models have tended to focus on responses associated with acute or short-term H. pylori infections (18, 19, 22, 24). In this study, we sought to evaluate host immune responses to H. pylori in a murine infection model. To this end, mice were infected with H. pylori SS1 and the humoral immune responses of the animals were assessed over time. The findings demonstrated that chronic H. pylori SS1 infection in mice induced humoral immune responses that closely mimicked those observed in human H. pylori infections. As has been found to be the case for infected humans, adaptive immune responses do not appear to be effective in eradicating an existent H. pylori infection in mice. This is the first report detailing the humoral responses of mice to a persistent H. pylori infection.

MATERIALS AND METHODS

Bacteria and growth conditions.

The mouse-adapted H. pylori strain SS1 was derived from a clinical isolate associated with upper abdominal pain and peptic ulcer disease, as described by Lee et al. (21). H. pylori SS1 was routinely subcultured on a blood agar (BA) medium (Blood Agar Base no. 2; Oxoid, Basingstoke, England) supplemented with 10% horse blood (bioMérieux, Marcy L’Etoile, France), containing a Helicobacter-selective antibiotic mixture (12), and incubated under microaerobic conditions at 37°C. Bacterial suspensions prepared from low-passage cultures of H. pylori SS1 (i.e., cultures that had undergone <10 passages in vitro), were stored at −80°C, in a tryptone casein soya broth solution (Pasteur Diagnostics) containing 25% glycerol.

Viable counts of H. pylori bacteria were made by a sloppy urea agar overlay technique. Briefly, samples to be tested were serially diluted in sterile 0.8% NaCl and then plated onto air-dried BA medium plates supplemented with 10 g of agar (Bacteriological Agar no. 1; Oxoid) per ml, 200 μg of bacitracin per ml, and 10 μg of naladixic acid (Sigma Chemical Co., St. Louis, Mo.) per ml (24). After 3 to 4 days of incubation, colonies with an H. pylori morphology were presumptively identified and enumerated. A sloppy urea overlay medium (composed of a modified Christensen’s urea solution [12] to which was added 0.6% Oxoid Agar no. 1) was applied to the plates (Fig. 1). After 5 to 10 min of incubation at room temperature, H. pylori colonies with urease-positive halos were enumerated.

FIG. 1.

FIG. 1

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Enumeration of H. pylori colonies from mouse gastric biopsies by a combined culture and urease activity technique. Homogenates of mouse gastric samples were serially diluted in saline and then used to inoculate serum plates (see Materials and Methods). After incubation, a sloppy urea overlay was applied to the plates, permitting the detection of urease-positive H. pylori colonies, which appear as fuchsia-colored zones on a yellow background.

Infection of mice with H. pylori SS1.

Six-week-old specific-pathogen-free Swiss, BALB/c, and C57BL/6 mice (Centre d’Elevage R. Janvier, Le-Genest-St-Isle, France) were housed in polycarbonate cages in isolators and fed a commercial pellet diet with water ad libitum. All animal experimentation was performed in accordance with institutional guidelines. Unless stated otherwise, Swiss mice were used for animal infection studies. Mice (n = 50) were inoculated intragastrically with suspensions of low-passage H. pylori SS1 cells, which had been harvested directly from 36- to 48-h plate cultures into peptone trypsin broth (Organotéchnique, La Courneuve, France). Each animal was administered a single 100-μl aliquot of the inoculating suspension by using polyethylene catheters (Biotrol, Paris, France) attached to 1.0-ml disposable syringes. A control group of mice (n = 10) was given peptone trypsin broth alone. At the appropriate times, mice were sacrificed and the saliva, gastric contents, sera, and stomachs of the animals were collected.

The infectious dose of H. pylori SS1 required to infect 100% of mice (ID100) was determined by orogastrically inoculating animals with suspensions containing known quantities of bacteria. The animals were sacrificed at 1 month postinoculation (p.i.).

Collection of saliva, gastric lavage, and serum samples.

To induce salivation, mice were given an intraperitoneal injection of 100 μg of pilocarpine (Sigma) prior to sacrifice. Salivary secretions were aspirated from the oral cavities of the mice and immediately stored at −20°C. Gastric secretions were collected by using a modification of the technique of Elson et al. (8), as described in detail elsewhere (13). Briefly, intact mouse stomachs were washed in phosphate-buffered saline (PBS) (pH 7.4) and then opened along the greater curvature to release the gastric contents directly into 2.7-ml aliquots of PBS in six-well tissue culture plates (Falcon; Becton Dickinson Labware, Franklin Lakes, N.J.). Solid matter present in the stomach contents of mice at the time of sacrifice was gently dislodged into the PBS solution by using a clean scalpel blade. To each sample of gastric contents was added 300 μl of EDTA (500 mmol per liter) and 3 μl each of the following protease inhibitors: leupeptin (2 mmol per liter) and pepstatin (2 mmol per liter) (Boehringer GmbH, Mannheim, Germany). The samples were centrifuged, and 20 μl of phenylmethylsulfonyl fluoride (100 mmol per liter) (Boehringer) and 100 μl of fetal calf serum (Gibco BRL, Cergy Pontoise, France) were added to each supernatant (2.0 ml) prior to storage at −20°C.

Serum was recovered in Sarstedt microtubes (Sarstedt France, Orsay, France) and stored at −20°C until tested.

Assessment of H. pylori infection in mice.

The presence of H. pylori infection in mice was determined by biopsy urease, quantitative culture, and histological analyses. Stomachs washed in PBS were placed on a flat surface (mucosal side up) and dissected longitudinally into three tissue fragments (so that each fragment contained cardia, body, and antrum). For each stomach, one fragment was immediately placed in urea-containing medium, another was placed in peptone trypsin broth (400 μl), and the remaining one was placed in formalin (2 ml). The presence of urease activity in tissue fragments was determined by monitoring biopsy urea agar (12) or urea-broth preparations (20) for ≤24 h at room temperature. To perform quantitative bacterial cultures on stomach samples, tissue fragments were homogenized in peptone trypsin broth with disposable plastic grinders and tubes (PolyLabo, Strasbourg, France). The homogenates were serially diluted in sterile saline, and the H. pylori colonies were enumerated by the technique described above.

Detection of antibodies by ELISA.

Antigen-specific antibodies in samples were detected by a previously described enzyme-linked immunosorbent assay (ELISA) technique (13). Briefly, 96-well Maxisorp plates (Nunc, Kamstrup, Denmark) were coated with sonicated whole-cell extracts of H. pylori SS1 (25 μg of protein per well), prepared in carbonate-bicarbonate buffer (pH 9.5). Diluted saliva (1:50), gastric lavage (1:10), and serum (1:100) samples were added in 100-μl aliquots to coated microtiter wells. To allow for nonspecific antibody binding, samples were also added to uncoated wells. Bound H. pylori-specific immunoglobulins were detected with biotinylated goat anti-mouse antibodies (Amersham, Les Ulis, France) and streptavidin-peroxidase conjugate (Amersham). Immune complexes were detected by reaction with a solution containing 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma) and hydrogen peroxide. Optical density (OD) readings were done at 405 and 492 nm with an ELISA Multiskan RC plate reader (Labsystems, Helsinki, Finland). The readings for uncoated wells were subtracted from those of the respective test samples. Cutoff values for each antibody class and each antibody sample type were determined from the mean OD values ± 2 standard deviations (SD) for the corresponding samples from naive uninfected mice. Samples with OD readings greater than these cutoff values were considered positive for H. pylori-specific antibodies. The quantities (in micrograms) of IgG subclass antibodies in sera were determined by interpolation of standard curves derived from a purified murine polyclonal IgG antibody (Sigma).

Histological analyses of gastric tissue samples.

Gastric tissue samples were taken from mice infected with H. pylori (day 3 and weeks 4 and 16 p.i.; n = 10/group), as well as from naive uninfected animals (n = 10). Stored samples were cut in longitudinal sections (4 μm) and stained by the hematoxylin-eosin, Warthin-Starry silver stain, and Giemsa techniques. Examination of the tissue sections for the presence of H. pylori bacteria and histopathological lesions was performed blind (by M. Huerre). Bacterial colonization was defined as the presence of any H. pylori bacteria in sections stained by the Warthin-Starry and/or Giemsa technique. The presence of inflammatory cell infiltrates (polymorphonuclear and mononuclear cells), erosive lesions, edema, hyperplasia, and lymphoid follicle formation was assessed according to the Sydney system (25). Morphological changes were thus classified into four grades: none, mild, moderate, or severe.

Statistics.

Differences in the bacterial loads or antibody responses between groups of animals were determined by the Mann-Whitney U test (two-sided). Differences were considered significant for a P of ≤0.05.

RESULTS

H. pylori SS1 bacterial numbers in mice remain constant over time.

To evaluate the capacity of H. pylori to colonize and persist in mouse gastric mucosae, Swiss mice (n = 50) were infected with an H. pylori SS1 suspension containing 107 CFU. H. pylori infection was present in all inoculated mice at day 3 p.i., as well as throughout the remainder of the study (i.e., 16 weeks) (Fig. 2). Quantitative culture of gastric tissue samples revealed H. pylori bacterial loads in the mice ranging from 4 × 104 to 8 × 106 CFU/g of tissue (Fig. 2). Although there were large variations in the numbers of bacteria recovered from mice within each group, no significant differences were observed between the mean numbers of bacteria recovered from mice at the different time points.

FIG. 2.

FIG. 2

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The numbers of H. pylori bacteria in gastric biopsies from Swiss mice over time. Mice (n = 50) that had been inoculated with a suspension of H. pylori SS1 were divided into five groups (each containing 10 mice) that were sacrificed at different times. Noninfected control mice (n = 2) were also sacrificed at each time point. The numbers of CFU in homogenates of gastric samples were determined as described in the text. Each point represents the mean of two estimations for a single mouse. Horizontal bars represent the geometric means for mice (n = 10) sacrificed at each time point.

Humoral immune responses in mice infected by H. pylori SS1.

At 1 week p.i., anti-H. pylori SS1 IgM and IgG antibodies were detected in the sera of infected mice in 70% (7 of 10) and 80% (8 of 10) of animals, respectively (Fig. 3). IgG antibodies were the dominant antibody class present in the sera of chronically infected mice. Anti-H. pylori IgA antibodies in serum were detected in only 1 of 40 mice sacrificed between day 3 and week 4 p.i., yet all animals (n = 10) were IgA seropositive at 16 weeks p.i. (Fig. 3).

FIG. 3.

FIG. 3

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Anti-H. pylori antibodies in the sera of Swiss mice infected by H. pylori SS1. Specific IgM, IgG, and IgA class antibodies in the sera of H. pylori-infected mice (Fig. 1) were detected by ELISA. The results are presented as A405–492 readings which correspond to the values given by diluted serum samples (1:100). Each point represents the mean of triplicate determinations for a single mouse. Sera from noninfected control mice (n = 10) were also tested and gave OD values of ≤0.05 (data not shown). Horizontal bars represent the geometric means for mice (n = 10) sacrificed at each time point.

H. pylori-specific IgG and IgA antibodies first appeared in the gastric contents of infected mice at 4 weeks p.i. and were present in 30% (3 of 10) and 20% (2 of 10), respectively, of animals at this time (Fig. 4). By week 16 p.i., all mice (n = 10) had specific IgG and IgA antibodies in their gastric compartments and IgA in their saliva (Fig. 4 and 5, respectively). In contrast, H. pylori-specific IgM antibodies were not detected in secretions from either of these mucosal sites.

FIG. 4.

FIG. 4

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Anti-H. pylori antibodies in the gastric contents of Swiss mice infected by H. pylori SS1. Specific IgG and IgA class antibodies in the gastric contents of H. pylori-infected mice (Fig. 1) were detected by ELISA. The results are presented as described for Fig. 3, except that gastric content samples were diluted 1:10. Each point represents the mean of triplicate determinations for a single mouse. Samples from noninfected control mice (n = 10) were also tested and gave OD values of ≤0.05 (data not shown). Horizontal bars represent the geometric means for mice (n = 10) sacrificed at each time point.

FIG. 5.

FIG. 5

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Anti-H. pylori antibodies in the saliva of Swiss mice infected by H. pylori SS1. Specific IgA class antibodies in the saliva of H. pylori-infected mice (Fig. 1) were detected by ELISA. The results are presented as described for Fig. 3, except that saliva samples were diluted 1:50. Each point represents the mean of triplicate determinations for a single mouse. Saliva from noninfected control mice (n = 10) were also tested and gave OD values of ≤0.05 (data not shown). Horizontal bars represent the geometric means for mice (n = 10) sacrificed at each time point.

Histological analyses of Swiss mice infected by H. pylori SS1.

H. pylori infection induced comparatively modest cellular immune responses in the gastric mucosae of Swiss mice, with mild chronic gastritis observed in infected animals at 16 weeks p.i. (Fig. 6). Inflammatory lesions were in some cases accompanied by polymorphoneutrophil cell infiltrates (3 of 10 mice) and lymphoid follicle formation (4 of 10 mice) (Fig. 6). Pathological lesions such as microerosions, hyperplasia, gland atrophy, or odema were absent from the animals.

FIG. 6.

FIG. 6

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Hematoxylin and eosin-stained sections of mouse gastric mucosae from uninfected and infected mice at 16 weeks p.i. (A) Normal gastric mucosa. (B and C) Gastric mucosa from an H. pylori-infected mouse, showing a mild diffuse gastritis in the mucosal and submucosal regions of the tissue. Lymphocyte (B) and polymorphoneutrophil (C) cell infiltrates are indicated. (D) Gastric mucosa from another H. pylori-infected mouse, showing a submucosal lymphoid aggregate. Bars = 100 μm.

Analysis of silver-stained sections of mouse gastric mucosae demonstrated the predilection of H. pylori SS1 bacteria for the stomach antrum. Nevertheless, in heavily colonized animals, H. pylori bacteria were also present in the fundus (Fig. 7).

FIG. 7.

FIG. 7

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Warthin-Starry silver stain of mouse gastric mucosa showing the presence of H. pylori bacteria in gastric body tissue. (A) A region of dense colonization with H. pylori bacteria present in the gastric glands and pits of the tissue. The bacteria often appear as clumps (arrows). (B) High-power magnification of a gastric gland colonized by spiral-shaped bacteria (arrows). Bars = 10 μm.

Determination of the ID100 required to infect mice.

H. pylori SS1 inocula of various doses were prepared by serial dilution of a stock suspension. The numbers of CFU present in the bacterial inocula were determined by quantitative culture (see Materials and Methods). Swiss mice were each intragastrically inoculated with 0.1 ml of the given suspension. The presence of H. pylori infection in mice was assessed at 1 month p.i. by urease activity and culture determinations. With an SS1 dose of 1.0 × 102 CFU, only 2 of 10 mice were found to be infected, but with 2.0 × 103, 2.9 × 104, and 1.2 × 105 CFU, all 10 mice in each group were infected. Thus, it was demonstrated that 2.0 × 103 CFU of the organism was required to infect all of the animals.

Colonization of different mouse strains with H. pylori SS1.

To determine and compare the susceptibilities of different mouse strains to H. pylori SS1, groups of Swiss, BALB/c, and C57BL/6 mice (n = 10 per group) were each inoculated with 1.2 × 105 CFU of H. pylori SS1 (equivalent to 100 times the ID100 for Swiss mice). At 1 month p.i., 2 of 10 gastric samples from the BALB/c mice were positive by the urea-broth technique, versus 8 of 10 and 7 of 10 Swiss and C57BL/6 mice, respectively (Table 1). The quantitative culture results for the different mouse groups showed reduced bacterial loads for BALB/c mice; however, these did not significantly differ from those of the other host strains (Table 1). The discrepancy between the urease and culture data for BALB/c mice was attributed to the relatively small size of the stomachs of the animals and the fact that H. pylori SS1 is restricted to the antrum in this host, whereas in C57BL/6 (21) and Swiss mice, H. pylori bacteria are also found in the body mucosae.

TABLE 1.

Infection of different mouse strains with H. pylori SS1

Mouse strain No. of mice positive/totala
Log CFU/g of tissueb,c Concn in serum (μg)c,d
Urease Culture IgG1 IgG2a
Swiss 8/10 10/10 5.69 ± 0.50* 0.28 ± 0.36† 0.19 ± 0.15‡
BALB/c 2/10 10/10 5.28 ± 0.82 0.60 ± 0.38 1.19 ± 0.27
C57BL/6 7/10 10/10 5.64 ± 0.59* 0.29 ± 0.22* 0.05 ± 0.04‡
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a

Mice each received 1.2 × 105 CFU of H. pylori SS1. The presence of H. pylori infection in gastric biopsies was assessed at 1 month p.i. by urease activity and culture. 

b

The numbers of H. pylori CFU recovered following homogenization of gastric biopsy samples. The results are presented as geometric means for 10 mice per group ± SD (for duplicate determinations). 

c

∗, P ≥ 0.05 versus BALB/c mice; † and ‡, P = 0.014 and 0.0002, respectively, versus BALB/c mice. 

d

The quantities of anti-H. pylori IgG subclass antibodies in the sera of infected mice were determined by ELISA. Results are the means ± SD for triplicate determinations per mouse (n = 10/group). 

Analysis of the quantities of H. pylori-specific IgG subclass antibodies observed in the sera of mice revealed differences between the various host strains. Notably, BALB/c mice had significantly larger quantities of IgG1 and IgG2a antibodies in serum than did the Swiss mice (P = 0.014 and 0.0002, respectively) and had more IgG2a antibodies than C57BL/6 animals (P = 0.0002) (Table 1). Moreover, in BALB/c mice, serum IgG2a antibodies appeared to be more abundant than those of the IgG1 subclass (Table 1).

Detection of H. pylori infection in mice.

Culturing of mouse gastric tissue was the most sensitive method for detecting H. pylori SS1 infection in mice, followed by histology (sensitivity = 73% [22 of 30 mice]) and the urea broth test (71% [37 of 52 mice]). The biopsy urea agar assay (44% [22 of 50 mice]) was found to be the least sensitive method of detection.

DISCUSSION

Lee and colleagues (21) reported that different mouse strains (C57BL/6, BALB/c, DBA/2, and C3H/He) could be infected to a high degree with a mouse-adapted H. pylori isolate (strain SS1). Moreover, they found that the levels of H. pylori colonization and bacterium-induced pathology in the mice varied markedly between the different host strains. The study presented here provides data which both complement and extend those reported previously. First, we determined the infectious dose required to infect Swiss mice and demonstrated that 103 CFU of H. pylori SS1 was sufficient to infect this host. Once infected, the mice harbored large numbers of bacteria, ranging from 105 to 107 CFU per g of gastric tissue, the levels of which remained constant throughout the study period (16 weeks). Second, and more importantly, we characterized and assessed the local and systemic immune responses of H. pylori-infected mice and showed similarities between these responses and those described for human infections.

To date, animal model studies have tended to focus on the histopathological lesions associated with H. pylori infections. In contrast, comparatively few studies have sought to determine the kinetics of host humoral immune responses to H. pylori infection. A key aim of the present investigation was to characterize the humoral immune responses of mice during the course of a persistent infection with H. pylori SS1. We found that H. pylori-infected mice produced strong antibody responses to the organism, with serum IgM antibodies detected in 7 of 10 animals within the first 7 days following infection (Fig. 3). Thereafter, H. pylori-specific IgG antibodies were the dominant class of immunoglobulin present in the sera of mice. No correlation between the presence of antibodies and the grade of H. pylori colonization in the animals was observed (11). Transient IgM responses, followed by IgG seroconversion, have previously been noted both in rodents that were experimentally infected with Helicobacter felis, a Helicobacter species of feline origin (15, 16), and in cases of acute H. pylori infection in humans (26, 29, 33). From serological studies of H. pylori-infected individuals, it was found that serum IgM antibodies could be detected between 14 and 30 days after infection (26, 33), while IgG antibodies appeared later (between 30 and 74 days p.i. [26, 33]).

Analysis of the local immune responses in H. pylori-infected mice demonstrated significant increases in the levels of gastric IgA and IgG antibodies in mice at 16 weeks p.i. (Fig. 4). Concurrently, IgA antibodies, but not IgG or IgM, were detected in the salivary compartments of infected mice (Fig. 5). It thus appears that H. pylori infection in mice preferentially induces the synthesis of gastric IgA and IgG antibodies in this host. Similar results have been obtained with other hosts, including mice experimentally infected with H. felis (16), as well as H. pylori-infected cats (17) and humans (6, 31, 33). On the other hand, the absence of mucosal IgM antibodies in H. pylori-infected mice contradicts the findings of clinical investigations (31, 33). The significance of this observation remains to be elucidated.

It was previously shown that antigen-specific antibodies present in the gastric secretions of H. felis-infected mice correlated with the proliferation of antibody-secreting plasmocytes in the gastric tissues of the animals (13, 30). Although the source of the anti-H. pylori gastric antibodies was not examined in the current investigation, there is indirect evidence to suggest that these antibodies may have been secreted by antibody-containing plasmocyte cells present within the gastric mucosa of the animals, rather than being the product of passive transudation of serum antibodies across the gastric mucosa. For one, if the antibodies were serum derived, then gastric IgG antibodies should have been detected in all animals at week 4 p.i., as this coincided with the time point at which serum IgG levels peaked (Fig. 3 and 4). Only three mice, however, had gastric IgG antibodies at this time, while all had them at week 16 p.i. Moreover, the presence of specific antibodies in the gastric secretions of H. pylori-infected mice appeared to coincide with the time at which the most significant amount of inflammation was observed in the animals. By performing IgG isotype analyses on sera and gastric contents, it should be possible to confirm the local origin of antibodies detected in the gastric secretions of H. pylori-infected mice.

Mild inflammatory lesions, occasionally accompanied by lymphoid follicle formation, were observed in mice infected with H. pylori SS1 (Fig. 6). These lesions were considerably less severe than those seen in H. felis-infected mice, while the lymphoid follicles in H. pylori-infected mice were smaller and less invasive than those seen in the former (11). These findings are consistent with the work of Lee et al. (21), who observed a mild gastritis in the antrum and body of H. pylori-infected mice at 3.5 months p.i., progressing to more severe inflammation in animals infected with the bacterium for 8 months. One explanation for the relatively modest inflammatory responses reported here might be that the Swiss mice were poorly colonized by H. pylori SS1, in which case the host immune system would need more time to respond to the infection. Certainly, lower densities of H. pylori bacteria were visible in histological sections taken from these animals than in equivalent sections from mice infected by H. felis (11). Quantitative culturing of gastric tissue homogenates from H. pylori-infected Swiss mice, however, revealed high bacterial loads in the animals, varying between 105 and 107 CFU/g of tissue (Fig. 2). Such bacterial densities compare favorably with those reported to be present in gastric biopsies from H. pylori-infected humans (1).

Another possible explanation for the mild gastritis observed in H. pylori-infected mice is the absence in this host of certain pathways involved in the induction of inflammatory responses to H. pylori in the human gastric mucosa. In humans, the proinflammatory cytokine interleukin-8 (IL-8) has been identified as playing an integral role in the recruitment and activation of inflammatory cells in the gastric mucosae of H. pylori-infected individuals (4, 6). Mice do not produce a homolog of human IL-8 (3), yet they do have a chemokine (named MIP-2) that has biological activities similar to those of IL-8. Whether this chemokine plays a role analogous to that of IL-8 in the induction of Helicobacter-related gastritis in the murine host has not been investigated. Nevertheless, since mice infected with H. felis develop severe chronic inflammatory lesions (9, 28, 32), it is unlikely that differences in proinflammatory cytokine production between animal host species can satisfactorily explain the mild gastritis seen in mice infected with H. pylori SS1.

Differences between H. pylori isolates have been cited as contributing to the variable severity of pathological and inflammatory lesions observed in mice infected with H. pylori (22). In particular, severe pathology in mice was reported to be associated with the expression in H. pylori strains of an immunodominant antigen (CagA) and a vacuolating cytotoxin (VacA) (22). Given that H. pylori SS1 expresses both of these proteins (21), expression of CagA and VacA per se does not appear to be sufficient to induce severe pathology in mice. On the basis of studies with murine Helicobacter models, it has been proposed that host factors, and not bacterial strain differences, are the major factors contributing to the pathology associated with H. pylori infection (28, 32). Indeed, host strain-dependent gastritis was demonstrated in mice that had been infected with either H. pylori (32) or H. felis (28, 32). Interestingly, we found that quantitative and qualitative differences between the humoral responses of different mouse strains infected with the same strain of H. pylori could also be observed (Table 1). The importance of host factors in the modulation of immune responses to H. pylori infection is further emphasized by anecdotal and experimental evidence demonstrating that some hosts are naturally more resistant to acute H. pylori infection (2, 7).

The findings presented here suggest that the H. pylori SS1 model is able to reproduce certain characteristics of human H. pylori infections. Among the advantages of this model is the capacity to study host immune responses during both the acute and the chronic stages of H. pylori infection. More importantly, it should be possible to use the H. pylori SS1 model to investigate the role of host factors in resistance to H. pylori infection.

ACKNOWLEDGMENTS

We thank Nicole Wuscher and Sabine Maurin for their technical assistance with the processing of histological samples.

Financial support was provided in part by Pasteur-Mérieux-Connaught (Lyon, France) and OraVax Inc. (Boston, Mass.).

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