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Infection and Immunity, May 1999, p. 2531-2539, Vol. 67, No. 5
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Local Secretory Immunoglobulin A and Postimmunization Gastritis
Correlate with Protection against Helicobacter pylori
Infection after Oral Vaccination of Mice
Takayuki
Goto,1
Akira
Nishizono,2,*
Toshio
Fujioka,1
Junko
Ikewaki,3
Kumato
Mifune,3 and
Masaru
Nasu1
Second Department of Internal
Medicine,1 Department of Infectious
Disease Control,2 and Department of
Microbiology,3 Oita Medical University,
Hasama-machi, Oita, Japan
Received 19 August 1998/Returned for modification 10 November
1998/Accepted 20 January 1999
 |
ABSTRACT |
C57BL/6 mice were orally immunized with five weekly doses of 2 mg,
200 µg, or 2 µg of Helicobacter pylori (Sydney strain) whole-cell sonicate combined with cholera toxin. One week after the
last vaccination, mice were challenged with 5 × 107
CFU of live H. pylori three times at 2-day intervals. At 6 or 18 weeks after the challenge, mice were sacrificed and bacterial cultures and histological studies of the stomach were performed. Vaccination with 2 mg/session or 200 µg/session inhibited H. pylori colonization by 90 and 100%, respectively. These mice
were considered protected. Lower levels of H. pylori-specific immunoglobulin A (IgA) were detected in fecal and
saliva samples before challenge. However, a significant increase in IgA
secretion in mucosal tissue and a higher labeling index for
IgA-positive lumina of pyloric glands were noted in these mice in
response to challenge and in a vaccine dose-dependent manner. In
protected mice, however, severe gastritis characterized by marked
infiltration of inflammation mononuclear cells was noted at 6 weeks
after challenge, compared with the gastritis seen in unprotected mice
or nonvaccinated, ordinarily infected mice. Marked expression of gamma
interferon mRNA was detected in the stomach of all protected mice, and
50% of these mice expressed interleukin 4 (IL-4) or IL-5 mRNA. Our findings suggest that local secretory IgA antibody and severe postimmunization gastritis correlate well with protection of mice against H. pylori infection.
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INTRODUCTION |
Chronic infection caused by
Helicobacter pylori is thought to be associated with chronic
active gastritis, peptic ulcer, and gastric malignancies, such as
mucosa-associated B-cell lymphoma and adenocarcinoma (2, 3, 21,
22). Furthermore, this organism was recently categorized as a
class I carcinogen by the World Health Organization (6), and
direct evidence of carcinogenesis was recently demonstrated in an
animal model (19). Although serological studies have
demonstrated H. pylori infection in approximately half of
the world's population, it is still not clear how H. pylori can induce such long-term infection.
Eradication of chronic H. pylori infection with antibiotics
markedly alters the natural history of gastroduodenal diseases and
reduces clinical symptoms. However, there are several problems with
antimicrobial therapy, such as side effects related to the use of a
high dose of antibiotics and the emergence of resistant strains of
H. pylori (11). Therefore, the development of a
prophylactic vaccine might be an attractive strategy against H. pylori infection, especially in young children.
Using a variety of animal models, several investigators have reported
that the use of a prophylactic vaccine of crude or purified H. pylori antigen induces protective immune responses against infection with Helicobacter species (1, 8, 10, 13,
16). The Helicobacter felis model in mice has provided
several important data. First, the use of a whole-cell sonicate or
recombinant protein as an oral immunogen prevents H. felis
colonization (10, 16). Second, infection with H. felis induces antigen-specific cellular immune responses
manifested by type 1 helper T (Th1) cells, although a type 2 helper T
(Th2)-cell response is also involved in protection against H. felis challenge (13).
In the present study, we attempted to delineate the possible mechanisms
of protection induced against H. pylori by oral vaccination with a mouse model of H. pylori infection.
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MATERIALS AND METHODS |
Animals.
Specific-pathogen-free 6-week-old female C57BL/6
mice were obtained from Seac Yoshitomi (Fukuoka, Japan). Mice were
housed in a specific-pathogen-free environment and were provided with free access to food and water. Experiments were performed according to
the guidelines of the Ethical Committee for Animal Experiments at Oita
Medical University, Oita, Japan.
Bacterial strain and preparation of H. pylori
antigen.
The Sydney strain of H. pylori, which was
kindly provided by A. Lee (School of Microbiology and Immunology,
University of New South Wales, Sydney, New South Wales, Australia)
(9), was grown in brucella broth containing 10% horse serum
under microaerobic conditions (5% O2, 10%
CO2, 85% N2) at 37°C. H. pylori
sonicated antigen was prepared as described previously (16).
Briefly, cultures were centrifuged at 1,000 × g for 10 min. The pellet was washed in phosphate-buffered saline (PBS), and
cells were disrupted by sonication. After centrifugation at 1,000 × g for 10 min, the supernatant was collected, the protein
concentration was determined, and the supernatant was frozen at
80°C until use.
Vaccination and challenge of mice.
Mice were divided into
four groups: groups 1 to 3, oral vaccination with cholera toxin (5 µg) as an adjuvant and H. pylori whole-cell sonicate at a
dose of 2 mg (group 1, n = 20), 200 µg (group 2, n = 20), or 2 µg (group 3, n = 20),
respectively (vaccinated/challenged groups); and group 4 (n = 20), ordinary infection without vaccination (nonvaccinated/infected group). Vaccination was repeated at weekly intervals for 5 weeks with the same doses of H. pylori as
those listed above. One week after the last vaccination, blood, fecal, and saliva samples were collected to monitor the immune response and
were stored at 80°C until use. One week after the last vaccination, all mice were challenged with 0.5 ml of live H. pylori
(5 × 107 CFU/ml) three times at 2-day intervals.
Assessment of H. pylori in gastric tissue.
At 6 or 18 weeks after the last challenge, all mice were sacrificed and the
stomach was isolated for examination for H. pylori, histological examination, and determination of cytokine expression by
reverse transcription (RT)-PCR. The stomach was washed in sterile 0.8%
NaCl and cut longitudinally into two pieces. One half was used for
bacterial examination, while the other was used for histological examination and determination of cytokine expression.
Examination for H. pylori.
Immediately after
homogenization of the stomach specimens, they were smeared on 7% sheep
blood agar (basic medium, Mueller-Hinton agar; BBL Microbiology
Systems, Cockeysville, Md.) and Belo-Horizonte medium and incubated at
37°C for 4 days under microaerobic conditions. The presence of
H. pylori in gastric tissue sections was also examined after
Gram staining.
Histological examination of gastric mucosa.
Longitudinal
sections of gastric tissues from the esophageal-cardiac junction to the
duodenum were fixed with neutral buffered 10% formalin and embedded in
paraffin. Five-micrometer sections were stained with hematoxylin-eosin
(HE) and Giemsa stains. Gastric sections were examined in a blinded
fashion by two independent examiners, who provided an assessment of the
overall grade of inflammation (on a scale of 0 to 7), which was
expressed as a sum of the overall intensity and extent of inflammation.
The intensity of inflammation was scored on a scale of 0 to 3 based on
criteria modified slightly from those described by Mohammadi et al.
(13): grade 0, rare inflammatory cells; grade 1, mild; grade
2, moderate; and grade 3, severe. The extent of inflammation was scored
on a scale of 0 to 4 based on the percentage of inflammatory cell infiltration of the mucosal surface (13): grade 0, none;
grade 1, <25%; grade 2, 25 to 50%; grade 3, 50 to 75%; and grade 4, >75%.
Immunohistochemical staining with secretory IgA antibody in
gastric tissue.
For immunohistochemical studies, 5-µm-thick
paraffin sections were preincubated with normal goat serum diluted 1:9
for 10 min at room temperature. Sections were then stained with rabbit anti-mouse immunoglobulin A (IgA) antibody (Zymed Laboratories Inc.,
South San Francisco, Calif.) for 20 min at room temperature, followed
by the avidin-biotin complex staining method (5). Sections
were reacted with 0.05 mol of Tris-HCl buffer per liter containing
0.02% 3,3'-diaminobenzidine tetrahydrochloride (Wako Pure Chemicals,
Osaka, Japan) and 0.005% H2O2, and the nuclei were counterstained with hematoxylin. Control sections incubated with
normal rabbit IgG instead of the primary antibody did not show
nonspecific staining. The labeling index (LI) for IgA-positive lumina
of pyloric glands was determined by examination of 500 glands in
sections of pyloric mucosa, and the percentage of lumina labeled with
anti-mouse IgA antibody was used for analysis.
Determination of antibody levels against H. pylori
antigens in serum, feces, and saliva.
Blood samples were obtained
from the tail vein at 7 days after the last immunization or by cardiac
puncture at 6 weeks after the last challenge. Secretory IgA antibody in
stool specimens was extracted from fecal pellets by incubation of the
samples with PBS containing 5% nonfat dry milk to yield approximately a 7% emulsion, 1 µg of aprotinin per ml, and 10 µM leupeptin
(Wako) (16). After extensive vortexing, the fecal material
was centrifuged at 13,000 × g for 10 min, and the
supernatants were used for the determination of IgA antibody. Saliva
samples were collected with a micropipette after intraperitoneal
injection of 200 µg of pilocarpine (Sigma) in sterile PBS.
Determination of IgG and IgA antibodies in blood, feces, and saliva
samples was performed by an enzyme-linked immunosorbent assay (ELISA).
Sera were tested for IgG at a dilution of 1:100 and for IgG1 and IgG2a
at a dilution of 1:25. Saliva and fecal samples were tested for IgA at
a dilution of 1:4 and undiluted, respectively. Microtiter plates (Nunc,
Roskilde, Denmark) were first coated with 100 µl of antigen (H. pylori whole sonicated antigen, 1 µg/well) in carbonate buffer
(pH 9.6) for 1 h at 37°C. They were then incubated with PBS
containing 5% bovine serum albumin (fraction V; Sigma Chemical Co.,
St. Louis, Mo.) for 1 h at 37°C to block nonspecific binding and
washed with PBS-Tween 20. Wells containing 100 µl of each test sample
were incubated for 1 h at 37°C. After being washed, the wells
were incubated with 100 µl of peroxidase-conjugated goat anti-mouse
IgG, goat-anti mouse IgG1, goat-anti mouse IgG2a, or goat-anti mouse
IgA antibodies (Cappel, Malvern, Pa.) for 1 h at 37°C. After the
wells were washed, conjugated peroxidase was detected with
2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) and
hydrogen peroxide. The absorbance at 414 nm was measured after 30 min
of incubation at room temperature. Each sample was tested in duplicate.
Extraction of RNA and RT-PCR.
To examine the expression of
cytokines in the stomach at the mRNA level, total RNA was extracted
from the stomach homogenate by the acid guanidium
thiocyanate-phenol-chloroform method (Nippon Gene Co., Tokyo, Japan) as
described previously (14). For this purpose, 1 µg of total
RNA was reverse transcribed in a final volume of 50 µl containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 5 mM
dithiothreitol, 500 µM deoxynucleoside triphosphate, 1 µM
oligo(dT)15 primer, 20 U of RNase inhibitor (Toyobo
Biomedicals, Osaka, Japan), and 500 U of Moloney murine leukemia virus
reverse transcriptase (Gibco-BRL, Gaitherburg, Md.). The reaction
mixtures were incubated for 2 h at 37°C, and the resultant cDNAs
were used for PCRs.
One microliter of cDNA was mixed in a final volume of 50 µl
containing 50 mM KCl; 10 mM Tris-HCl (pH 8.8); 1.5 mM
MgCl
2; 0.1%
Triton X-100; 200 µM each dATP, dCTP, dGTP,
and dTTP; 20 µM each
primer (see below); and 0.2 U of
Taq
DNA polymerase (Takara Shuzo,
Kyoto, Japan). Amplification was
performed for 35 cycles in a
DNA thermal cycler (ASTEC Co., Fukuoka,
Japan) as follows: 94°C
for 1 min, 55°C for 2 min, and 72°C for 1 min. The PCR products
were electrophoresed on a 2% agarose gel,
stained with ethidium
bromide, and observed with a UV transilluminator.
The primer sequences
were as follows (
4,
7,
15,
20): gamma
interferon (IFN-
)
sense primer, 5'-AACGCTACACACTGCATCT-3';
IFN-
antisense primer,
5'-TGCTCATTGTAATGCTTGG-3';
interleukin 4 (IL-4) sense primer,
5'-TAGTTGTCATCCTGCTCTT-3'; IL-4 antisense primer,
5'-CTACGAGTAATCCATTTGC-3';
IL-5 sense primer,
5'-AAGATGCTTCTGCACTTGA-3'; IL-5 antisense primer,
5'-ACACCAAGGAACTCTTGCA-3';
-actin sense primer,
5'-ATGGATGACGATATCGCT-3';
and
-actin antisense primer,
5'-ATGAGGTAGTCTGTCAGGT-3'.
For semiquantitative determination of the expression of cytokine mRNA,
we constructed standard recombinant plasmids containing
a partial
genome of IFN-
, IL-4, IL-5, or
-actin. The cloning
procedures
were essentially similar to those described by Sambrook
et al.
(
17). Using cDNA obtained from the thymus gland of newborn
mice, we amplified target signals of cytokines by using the specific
primers described above. The amplified products were inserted
into a
pGEM-T vector (Promega Co., Madison, Wis.) and subcloned
by
transformation with
Escherichia coli JM109 competent cells
(Toyobo). The concentration of cloned recombinant plasmid containing
each cytokine DNA was estimated by measurement of the absorbance
at 260 nm. Tested samples were amplified by PCR in parallel with
10-fold
serially diluted standard recombinant plasmid. The copy
number of each
sample was determined by comparing the density
with that of standard
cDNA by agarose gel electrophoresis and
ethidium bromide staining.
There were no significant differences
among the samples with regard to
the expression of
-actin
mRNA.
Statistical analysis.
Differences in H. pylori-specific IgG and IgA antibody levels, grade of gastric
inflammation, and LI for IgA-positive lumina of pyloric glands among
experimental groups were examined for statistical significance by
analysis of variance or Student's t test. Differences in
the rate of protection against H. pylori infection were
analyzed by Fisher's exact probability test. A P value of
<0.05 was considered statistically significant.
| |
RESULTS |
H. pylori-specific antibody levels in feces, saliva,
and sera obtained from orally vaccinated mice.
Blood, fecal, and
saliva samples were collected, and the antibody responses against
H. pylori antigens were examined by an ELISA 1 week after
the last vaccination. Although the antibody levels were low and the
biologic significance was undefined, increases in fecal and saliva IgA
antibody levels were observed in vaccinated (group 1 and 2) (Fig.
1A) mice and in group 1 mice (Fig. 1B), respectively, compared with the nonvaccinated controls (P,
<0.05). In the vaccinated groups, the levels of serum H. pylori-specific IgA and IgG antibodies were elevated in proportion
to the doses of H. pylori whole-cell sonicate (Fig. 1C and
D).
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FIG. 1.
Detection of antibody responses in the feces, saliva,
and sera of vaccinated (open bars) and nonvaccinated (filled bars) mice
at 1 week after the last vaccination. H. pylori-specific
fecal (with undiluted extract) IgA (A), salivary (at a 1:4 dilution)
IgA (B), and serum (at a 1:100 dilution) IgA (C) and IgG (D) antibodies
were measured by an ELISA. Data represent the mean ± standard
deviation optical density at 414 nm for each group (n, 17 to
20 mice). P values were <0.05 (*), <0.01 (**), and
<0.005 (***) compared with the values for the corresponding
nonvaccinated mice (Student's t test).
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Effect of vaccination against H. pylori infection.
The effect of vaccination was based upon protection against live
H. pylori challenge infection. Protection was judged by the absence of H. pylori, determined by both culturing for
H. pylori and light microscopic examination of gastric
sections stained with Giemsa stain at 6 and 18 weeks after challenge.
In nonvaccinated/infected mice, H. pylori had colonized well
at 6 and 18 weeks after the challenge. The percentages of protection
were 90 and 100% in vaccinated/challenged groups 1 and 2, respectively, at 6 weeks after the challenge. In contrast, in 8 of 10 mice in group 3, H. pylori grew in the stomach, indicating
that these mice were not protected. The difference in the levels of
H. pylori colonization between vaccinated/challenged mice (groups 1 and 2) and nonvaccinated/infected mice was
statistically significant (P, <0.001) (Fig.
2).
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FIG. 2.
Effect of oral vaccination with 5 µg of cholera toxin
and 2 mg (group 1), 200 µg (group 2), or 2 µg (group 3) of H. pylori whole-cell sonicate on H. pylori infection.
Protection against H. pylori infection was based on the
absence of H. pylori, determined by both bacterial culturing
and light microscopic examination of stomach sections (Giemsa stain) at
6 weeks after the last challenge (n, 10 mice). An asterisk
indicates that the P value was <0.001 compared with the
value for the corresponding nonvaccinated/infected mice (Fisher's
exact test).
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Michetti et al. (
12) reported that oral vaccination
occasionally induces gastritis without
H. pylori challenge.
Therefore,
we examined whether our oral vaccination alone induced
gastric
inflammation. Inflammatory cell infiltration was not observed
in the stomachs of all vaccinated mice at 6 weeks after the last
vaccination (data not shown). In nonvaccinated/infected mice,
however,
mild inflammation was present in the antral region at
6 weeks after
infection (Fig.
3A). In gastric tissue
sections
of these mice,
H. pylori was
found in the antral region (Fig.
3B). The cell infiltrates consisted of
diffuse aggregates of numerous
neutrophils, plasma cells, and a few
mononuclear cells. These
histopathological inflammatory features were
compatible with active
chronic gastritis.
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FIG. 3.
Micrographs of gastric tissues of
vaccinated/challenged mice and nonvaccinated/infected mice at 6 weeks
after challenge. (A) Antral mucosa of a representative
nonvaccinated/infected mouse, showing active chronic inflammation
mainly in the submucosal layer (HE stain; original magnification,
×125). (B) Large numbers of spiral bacteria throughout the antral
crypts in a representative nonvaccinated/infected mouse (Giemsa stain;
original magnification, ×1,000). (C) Fundic mucosa of a representative
protected mouse in the vaccinated/challenged group, showing high
intensity of infiltration by inflammatory cells composed mainly of
mononuclear cells (HE stain; original magnification, ×100).
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In vaccinated/challenged mice (groups 1 and 2) the grade of gastritis
was more severe than that in nonvaccinated/infected
mice; marked
infiltration of mononuclear cells was noted, mainly
in the corpus of
the stomach (Fig.
3C). In unprotected mice the
gastritis score at 6 weeks after the challenge was significantly
lower than that at 18 weeks
after the challenge in nonvaccinated/infected
mice (Fig.
4). On the other hand, the gastritis
score at 6 weeks
after the challenge in protected mice was
significantly higher
than that in unprotected mice (
P,
<0.0005). In addition, gastritis
observed in protected mice
lasted for 18 weeks, and the severity
at 6 weeks after the challenge
had reached levels almost equal
to those observed at 18 weeks for
unprotected or nonvaccinated/infected
mice.
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FIG. 4.
Gastritis scores in vaccinated/challenged and
nonvaccinated/infected mice at 6 (open bars) and 18 (filled bars) weeks
(W) after the challenge. HE-stained gastric sections were scored based
on the overall grade of inflammation (on a scale of 0 to 7), which
included the intensity and extent of inflammation (see Materials and
Methods). Data represent the mean ± standard deviation gastritis
scores for each group (n, 5 to 10 mice). The asterisk
indicates that the P value was <0.0005 compared with the
value for the corresponding nonvaccinated/infected mice at 6 weeks
after the challenge (Student's t test).
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Immunohistopathological examination of gastric tissue.
To
examine whether secretory IgA is induced and actively secreted in
gastric mucosa following oral vaccination, immunohistochemical staining
for IgA antibody in gastric mucosa was performed at 6 weeks after the
challenge. The number of gastric gland lumina positively stained with
IgA antibody was significantly higher in vaccinated/challenged mice
(Fig. 5A) than in
nonvaccinated/infected mice (Fig. 5B) (P,
<0.05). In vaccinated/challenged mice, the LI for IgA-producing
glands in groups 1 and 2 was significantly higher than that in group 3 (P, <0.001) (Fig. 5C).
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FIG. 5.
Immunohistochemical analysis of gastric tissues from
vaccinated/challenged mice (magnification, ×100) (A) and
nonvaccinated/infected mice (magnification, ×100) (B) at 6 weeks after
the challenge. (C) IgA LI for the gastric mucosa in
vaccinated/challenged, nonvaccinated/infected, and normal control mice
at 6 weeks after the challenge. The LI represent the percentage of
anti-mouse IgA antibody-labeled lumina among 500 pyloric glands of the
gastric mucosa. Data represent the mean ± standard deviation LI
for each group (n, 10 mice). An asterisk indicates that the
P value was <0.05 compared with the value for the
corresponding nonvaccinated/infected mice at 6 weeks after the
challenge (Student's t test). A section sign indicates that
the P value was <0.001 compared with the value for the
corresponding vaccinated/challenged mice (2 µg) at 6 weeks after the
challenge (Student's t test).
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H. pylori-specific serum IgG antibody subclasses before
and after challenge.
To determine the phenotypes of Th cells
induced by oral vaccination, the subclasses of H. pylori-specific IgG were determined for vaccinated and/or infected
mice by an ELISA before and at 6 weeks after challenge. At 1 week after
the last vaccination, group 1 produced serum IgG1 antibody (P,
<0.05 [versus control mice]). At 6 weeks after the challenge,
the titer of serum IgG1 in protected mice was significantly higher than
that in unprotected mice (P, <0.05) (Fig.
6). Although these findings do not
reflect the local immune responses in the stomach, they suggest that
Th2 cell-mediated immune responses are operational at least in the whole body.
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FIG. 6.
Secondary responses of IgG subclasses in vaccinated mice
at 6 weeks after challenge infection. H. pylori-specific
serum (at a 1:25 dilution) IgG1 and IgG2a antibodies were quantitated
by an ELISA. Data represent the mean ± standard deviation optical
density at 414 nm for each group (n, 10 mice). The asterisk
indicates that the P value was <0.05 compared with the
value for the corresponding nonvaccinated mice at 1 week after the last
vaccination (Student's t test). The section sign indicates
that the P value was <0.05 compared with the value for the
corresponding unprotected mice in the vaccinated/challenged group and
for the nonvaccinated/infected mice at 6 weeks after the challenge
(Student's t test).
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Local expression of cytokine mRNAs after challenge.
To further
determine the local immune responses in the stomach, we compared the
expression of mRNAs of IFN-, IL-4, and IL-5 by RT-PCR using stomach
homogenates of vaccinated/challenged and nonvaccinated/infected mice
(Fig. 7). Interestingly, the expression of IFN- mRNA in the stomach was significantly higher in protected than in unprotected mice. In nonvaccinated/infected mice the level of
IFN- mRNA was lower than 102 copies and was lower than
that in vaccinated/challenged mice (Fig. 7). The level of IFN- mRNA
in vaccinated mice without challenge was undetectable at 6 weeks after
the last vaccination (data not shown). On the other hand, IL-4 mRNA was
detected in 3 of 10 protected mice and IL-5 mRNA was detected in
another 2 of 10 protected mice. In other words, 5 of 10 protected mice
expressed the mRNA of either IL-4 or IL-5, although the levels were
lower than 102 copies. The same mRNAs were not detected in
stomach homogenates of unprotected mice and nonvaccinated/infected mice
(Fig. 7).
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FIG. 7.
Expression of IFN-, IL-4, and IL-5 mRNAs in the
stomachs of nonvaccinated/infected and vaccinated/challenged mice at 6 weeks after the challenge. mRNAs were detected by RT-PCR and expressed
as the copy number determined semiquantitatively (see Materials and
Methods). Mice were vaccinated with 2 mg ( ), 200 µg ( ), and 2 µg ( ) of whole-cell sonicate;  , nonvaccinated/infected mice.
The ordinate is the logarithmic copy number of each cytokine mRNA.
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DISCUSSION |
Several investigators have recently suggested that oral
vaccination with either H. pylori whole-cell antigen or
purified antigen and appropriate adjuvants can prevent infection by
Helicobacter species (1, 8, 10, 12, 13, 16). In
the present study, we showed that oral vaccination with a dose of
greater than 200 µg of H. pylori whole-cell sonicate per
session for 5 weeks was effective in protecting C57BL/6 mice against
H. pylori infection.
We first noted a severe inflammatory cell infiltration in the corpus at
6 weeks after the challenge, especially in protected mice. We also
demonstrated that the development of more severe inflammation at this
stage might lead to protection. A number of investigators (1, 13,
16) have reported that gastric inflammation occurs in immunized
mice after challenge; this effect has been referred to as
postimmunization gastritis. However, the details of postimmunization
gastritis differ among studies. For example, Pappo et al.
(16) showed that oral immunization with recombinant urease
and cholera toxin resulted in protection against H. felis
infection and that the gastritis in recombinant urease-immunized mice
subsequently challenged with H. felis exhibited
Thy1.2+ T cells in mucosal and submucosal layers. On the
other hand, Ferrero et al. (1) demonstrated that orogastric
immunization of mice with recombinant H. pylori GroES-like
and urease B-subunit proteins protected against H. felis
challenge and that mild gastric inflammation may be a necessary
prerequisite for successful immunization. In that study, the severity
of postimmunization gastritis was milder than that of ordinary H. felis infection. Mohammadi et al. (13) showed that the
grade of postimmunization gastritis was more severe than that of
ordinary H. felis infection when H. felis
whole-cell sonicate was used as the oral immunogen, similar to our
findings. These results suggest that postimmunization gastritis might
correlate with protection against Helicobacter challenge. However, the extent of postimmunization gastritis is different in each
experimental design. The different natures of oral immunogens with
regard to immunogenicity or cross-reactivity against host proteins,
differences in mouse strains used, and the strain diversity of
Helicobacter species might be the factors responsible for
the differences in the extent of postimmunization gastritis. The same can be said of the discrepant finding that gastric inflammation was not
observed in all vaccinated mice without H. pylori challenge in the present study, in contrast to the occasional induction of
gastritis by vaccination only demonstrated by Michetti et al. (12).
Measurement of mRNAs for a group of cytokines in the stomach in the
present study showed that IFN- mRNA was strongly expressed in
protected mice. In contrast, the expression was weaker (less than
103 copies) in unprotected mice as well as in
nonvaccinated/infected mice. We also showed that the expression of the
mRNA for either IL-4 or IL-5 was detected in 50% of protected mice.
These findings suggest that postimmunization gastritis represents a
reactive inflammation of the gastric mucosa induced primarily by Th1
immune responses together with secondary Th2 responses induced by the challenge. In support of the involvement of Th1 immune responses or
IFN- in postimmunization gastritis, Mohammadi and coworkers (13) showed that treatment of nonimmunized/infected and
immunized/challenged mice with anti-IFN- antibody led to a
significant reduction in the severity of gastric inflammation.
With regard to mucosal immunity, it is clear that local IgA antibody
plays a primary role in protection against foreign organisms. However,
there are no reports directly indicating that gastric tissue functions
as an effector organ of mucosal immunity, similar to gut-associated
lymphoid tissue. In the present study, we observed protection against
H. pylori in vaccinated mice (mainly groups 1 and 2), in
which salivary and fecal H. pylori-specific IgA was detected. In these mice, high levels of IgA were also secreted in the
pyloric mucosa, and these levels were vaccine dose dependent (Fig. 5).
However, this IgA is not necessarily specific for H. pylori,
since it has been demonstrated that IFN- and IL-4 up-regulate the
expression of the polymetric immunoglobulin receptor, which transports
IgA in human intestinal epithelium (18). In support of the
involvement of Th2 cell-mediated immune responses in the stomach, we
demonstrated the expression of mRNAs for IL-4 and IL-5 in the stomachs
of mice protected against challenge infection (Fig. 7). To our
knowledge, this is the first report of mRNAs for these cytokines in
situ, although a previous study demonstrated the secretion of these
cytokines in the supernatants of lymphocyte cultures from the stomach
after antigenic stimulation (13). Taken together, these
findings suggest that local secretory IgA antibody in gastric mucosal
tissue correlates well with protection against H. pylori
infection. However, whether the stomach functions as a primary priming
site for inducing the protective IgA response remains to be elucidated
in future studies.
Cellular immune responses to H. pylori have been studied for
humans, but the nature of such responses, including the possible involvement of protective immunity, is not well defined. In the present
study, we demonstrated that Th1 cell-mediated immune responses occur in
the stomach, as indicated by the marked expression of IFN- mRNA.
However, the mechanism(s) through which a challenge infection of
vaccinated mice induces the strong expression of IFN- is still not
clear, particularly in protected mice. The expression of IFN- might
be associated with the appearance of postimmunization gastritis
(13). Furthermore, the development of severe
postimmunization gastritis during the early postchallenge infection
period is probably an essential prerequisite for protection, as
demonstrated in the present study and previous reports (1, 13). However, the mechanism of protection after the appearance of
postimmunization gastritis remains unidentified.
Further studies are necessary to clarify these issues and identify the
antigen(s) that induces protective immunity following oral vaccination,
with the hope of preventing postimmunization gastritis.
| |
ACKNOWLEDGMENTS |
We thank Adrian Lee for donating the Sydney strain and for
careful reading of the manuscript and Kiyomi Ohno and Mami Kimoto for
technical assistance. We also thank F. G. Issa (Word-Medex, Sydney, Australia) for careful reading and editing of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Infectious Disease Control, Oita Medical University, Idaigaoka 1-1, Hasama-machi, Oita 879-5593, Japan. Phone: 81-97-586-5701. Fax:
81-97-586-5719. E-mail: a24zono{at}oita-med.ac.jp.
Editor:
R. N. Moore
| |
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Infection and Immunity, May 1999, p. 2531-2539, Vol. 67, No. 5
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
