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* Department of Clinical Immunology, University of Göteborg, Göteborg, Sweden;
Department of Immunology, AstraZeneca R&D Boston, Waltham, MA 02451; and
Department of Pathology, Örebro University Hospital, Örebro, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. However, IFN-, but not IL-12, was involved in the
development of gastritis because IFN--/- (GKO) mice
exhibited significantly less inflammation as compared with
IL-12-/- or wild-type (WT) mice. Both
IL-12-/- and GKO mice failed to develop protection
following oral immunization with H. pylori lysate and
cholera toxin adjuvant. By contrast, Th2-deficient,
IL-4-/-, and WT mice were equally well protected. Mucosal
immunization in the presence of coadministered rIL-12 in WT mice
increased Ag-specific IFN--producing T cells by 5-fold and gave an
additional 4-fold reduction in colonizing bacteria, confirming a key
role of Th1 cells in protection. Importantly, only protected
IL-4-/- and WT mice demonstrated substantial influx of
CD4+ T cells in the gastric mucosa. The extent of
inflammation in challenged IL-12-/- and GKO mice was much
reduced compared with that in WT mice, indicating that IFN-/Th1
cells also play a major role in postimmunization gastritis. Of note,
postimmunization gastritis in IL-4-/- mice was
significantly milder than WT mice, despite a similar level of
protection, indicating that immune protection is not directly linked to
the degree of gastric inflammation. Only protected mice had T cells
that produced high levels of IFN- to recall Ag, whereas both
protected and unprotected mice produced high levels of IL-13. We
conclude that IL-12 and Th1 responses are crucial for H.
pylori-specific protective immunity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The roles of Th1 or Th2 cells in Helicobacter-specific
protective immunity are, however, still incompletely understood.
Studies with H. felis infection have reported on a dominant
IFN- Th1-type response, which enhanced gastric inflammation without
reducing bacterial load (12). In adoptive transfer
studies, it was further shown that H. felis-specific Th1 and
Th2 cell lines enhanced gastric inflammation, but only Th2 cells were
associated with reduced bacterial colonization (13). Also,
repeated therapeutic oral immunizations in BALB/c mice stimulated
significant protection, which correlated with a gradual skewing of the
T cell response toward Th2 (14). However, both Th1 and Th2
cells generated after systemic immunization with H. felis
lysate and CFA or aluminum hydroxide, respectively, mediated protection
(15). Other studies have indicated that IL-4- and
IL-13-dependent Th2 type responses were, in fact, not required for
protection, as immunized IL-4R
-/- mice were
strongly protected against H. pylori infection
(16). Finally, a replication-defective adenovirus
infection, causing Th1 skewing, therapeutically reduced bacterial
colonization in chronically infected WT mice, but not in IFN-- or
IL-12-deficient mice (17).
Because of the inconsistencies in our current information on the
regulatory roles of Th1 and Th2 cells in protective immunity against
Helicobacter infection, we investigated this matter in some
detail using IL-12- (IL-12-/-), IFN-- (GKO),
or IL-4-deficient (IL-4-/-) mice and compared
the results to that found in WT mice. We found that only if Th1-type
responses dominated, as seen in the IL-4-/- and
WT mice following oral immunizations, did we observe protection against
a live challenge infection with H. pylori bacteria.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-12-deficient (IL-12-/-; Ref.
18), IFN--deficient (GKO; Ref. 19), and
IL-4-deficient (IL-4-/-; Ref. 20)
mice on a C57BL/6 background, were bred in ventilated cages under
pathogen-free conditions at the University of Göteborg Animal
Facility, Department of Medical Microbiology and Immunology
(Göteborg, Sweden). Wild-type (WT) C57BL/6 mice were obtained
from B&K Universal AB (Sollentuna, Sweden) or from Taconic Farms
(Germantown, NY). All mice were seronegative for
Helicobacter spp. Ags before infection or immunization. Age-
and sex-matched animals were used throughout the study.
Immunization
Groups of 7–10 mice were immunized orally with a blunt feeding needle (Popper & Sons, New Hyde Park, NY) four times at weekly intervals with 500 µg of H. pylori whole cell lysate Ags and 10 µg of CT adjuvant (List Biological Laboratories, Campbell, CA) as described elsewhere (11). For experiments assessing the role of IL-12, wild-type mice were immunized once by the intranasal route with 100 µg of H. pylori lysate plus 1 µg of CT adjuvant in 20 µl via a pipette tip. Control groups received H. pylori lysate alone, or CT adjuvant alone, as indicated. Murine rIL-12 (R&D Systems, Minneapolis, MN) at 1 µg per dose was administered orally in 50 µl, followed by live challenge infection with H. pylori bacteria.
H. pylori growth conditions and challenge
H. pylori SS1 was grown on tryptic soy agar (TSA)
plates (BD Biosciences, Cockeysville, MD) containing 5% sheep blood
and 100 µg of vancomycin, 3.3 µg of polymyxin B, 200 µg of
bacitracin, 10.7 µg of nalidixic acid, and 50 µg of amphotericin B
(Sigma-Aldrich, St. Louis, MO) per milliliter. The plates were
incubated for 48 h at 37°C under microaerobic conditions (10%
CO2, 5% O2, and 85%
N2). The bacteria were then harvested and
inoculated into Brucella broth (BD Biosciences)
supplemented with 5% heat-inactivated FCS (Biochrom, Berlin,
Germany). A gas mixture consisting of 5%
O2, 10% CO2, and 85%
N2 was injected directly into culture flasks
containing the bacteria before they were sealed and shaken at 120 rpm
at 37°C for 24 h. Cultures were grown to an
OD600 of 0.3 (
5 x
108 CFU/ml), and diluted in Brucella
broth for inoculation. Before use, H. pylori cells were
analyzed in wet mounts to assess motility and morphology, and subjected
to urease, catalase, and oxidase tests. To establish a primary H.
pylori infection, mice were challenged intragastrically with
3 x 108 CFU of H. pylori on two
consecutive days. To assess protection after immunization, the mice
were challenged with 5 x 107 CFU of
H. pylori 2 wk after the last immunization. Mice were killed
2 wk postchallenge, and the gastric tissue was processed for urease
activity, quantitative H. pylori culture, histopathology,
and immunohistochemical analyses as described below.
Preparation of H. pylori whole cell lysate Ags
H. pylori was grown on selective blood agar plates for 48 h at 37°C under microaerobic conditions (10% CO2, 5% O2, and 85% N2) and suspended in PBS. The cells were then washed three times in PBS by centrifugation at 6000 x g for 10 min at 4°C before being disrupted by freeze-pressing with X-press (21). Briefly, the bacterial suspensions were frozen at -35°C in the form of cylindrical rods, which fit into the pressure chamber and were extruded by a piston forced back and forth by means of a hydraulic pump. After centrifugation (6000 x g) to remove cell fragments, the preparation was filtered through a 0.2-µm membrane filter (Schleicher & Schuell, Dassel, Germany). The protein content was determined by the Bio-Rad protein assay (Hercules, CA), and aliquots were frozen at -70°C until used.
Gastric tissue analyses
The stomach was dissected along the greater curvature and
divided into four longitudinal strips for assessment of urease
activity, H. pylori culture, histopathology, or
immunohistochemical analyses. For histopathology, longitudinal segments
including the antrum and corpus plus a piece of attached intestine were
fixed in 4% neutral buffered formaldehyde, embedded in paraffin, and
sectioned at 3 µm by Histolab Products (Göteborg, Sweden). For
immunohistochemistry, gastric segments were placed into Histocon
(Histolab Products) at 4°C. The tissues were then placed in plastic
forms (Cryomold; Miles, Elkhart, IN) filled with OCT compound (Miles)
and were subsequently snap-frozen in isopenthane in liquid nitrogen
(N2) for 60 s. Frozen cross-sections (7 µm)
were prepared on microslides using a cryostat-1720 (Leitz, Wetzlar,
Germany) and frozen at -70°C.
Assessment of bacterial colonization
The presence of H. pylori in gastric tissue was assessed by urease activity measured spectrophotometrically at 550 nm using a colorimetric assay (11). For quantitative H. pylori culture of gastric tissue (11), longitudinal segments were homogenized in 0.5 ml of Brucella broth supplemented with 5% FCS, and replicate serial 10-fold dilutions were plated on Helicobacter-selective blood agar plates. The plates were incubated at 37°C under microaerobic conditions (10% CO2, 5% O2, and 85% N2), and the quantitation of the CFU was performed 7 days later. In the present study, protection against H. pylori infection was defined as a significant reduction in the colonizing bacteria in the stomach.
Histopathology
For evaluation of gastritis, H&E-stained sections were scored based on the degree of infiltrating lymphocytes, plasma cells, and neutrophils (22). Grades were defined as follows: 0, none; 1, a few leukocytes scattered in the deep mucosa; 2, moderate numbers of leukocytes in the deep to mid mucosa and occasional neutrophils in gastric glands (microabscesses); 3, dense infiltrates in the deep to mid mucosa, a few microabscesses, and one or two lymphoid aggregates; and 4, dense, diffuse infiltrates throughout the lamina propria and into the submucosa, frequent microabscesses, and prominent lymphoid aggregates.
Epithelial changes
For evaluation of epithelial changes, H&E-stained sections were scored based on the degree of epithelial destruction in the corpus including parietal cell loss and hyperplasia of the surface epithelium as described by Ermak et al. (23). Briefly, epithelial scores were defined as follows: 0, none; 1, small, focal areas of parietal cell loss in the corpus and/or hyperplasia of the surface epithelium; 2, epithelial changes throughout 75% of the mucosa; 3, epithelial changes throughout the mucosa plus one to three microabscesses or cystic glands; or 4, epithelial changes throughout the mucosa plus four or more microabscesses or cystic glands.
Immunohistochemistry of gastric tissue
Frozen sections from gastric tissue were fixed in 50% acetone for 30s followed by 100% acetone for 5 min at 4°C. After washing in PBS, sections were blocked with avidin-biotin blocking reagents (Vector Laboratories, Burlingame, CA) and endogenous peroxidase activity blocked with 0.3% H2O2 in PBS. Sections were then incubated with 5% horse serum in 0.1% BSA (Sigma-Aldrich)/PBS for 15 min in a humid chamber. Sections were incubated with biotinylated rat mAbs against mouse CD4 or CD8 (BD PharMingen, San Diego, CA). For detection of CD3 cells, sections were incubated with rat mAb against mouse CD3 (BD PharMingen) followed by incubation with biotinylated rabbit anti-rat IgG (H + L; Vector Laboratories). Sections were then incubated with HRP-conjugated to an avidin-biotin complex (ABC-Elite kit; DAKO, Glostrup, Denmark). Cell-bound peroxidase was detected with 3-amino-9-etylcarbazole (AEC) (Vector Laboratories) and H2O2. Sections were counterstained with hematoxylin. Control sections were incubated without specific mAb. Stained sections were scored from 0 to 4 based on the extent of infiltrating T cells. Scores were defined as follows: 0, none; 1, a few T cells scattered in the mucosa; 2, moderate numbers of T cells in the gastric mucosa; 3, dense infiltrates in the deep to mid mucosa; 4, dense, diffuse infiltrates throughout the lamina propria and into the submucosa.
Serum and gastric samples
Blood was obtained from the axillary plexus of the mouse at sacrifice. Gastric secretions were collected with absorbent wicks positioned longitudinally in the gastric lumen (24), after extensive rinsing with PBS containing 0.2 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (Calbiochem, La Jolla, CA), 1 µg of aprotinin per ml, 10 mM leupeptin (Sigma-Aldrich), and 3.25 µM Bestatin (Boehringer Mannheim Biochemicals, Indianapolis, IN) protease inhibitors. For extraction of gastric secretions from the wick, 0.5 ml of protease inhibitor containing 5% nonfat dry milk was added to each sample tube containing two wicks, vortexed extensively, and then frozen at -70°C until analyzed.
Serum and gastric anti-H. pylori Ab determinations by ELISA
Flat-bottom 96-well microtiter plates (Nunc, Roskilde, Denmark) were incubated with 10 µg/ml H. pylori lysate in PBS. After washing and blocking with PBS containing 0.1% BSA, the wells were incubated with serial dilutions of sera or gastric secretions. The wells were then incubated with the appropriate dilutions of alkaline phosphatase (AP)-conjugated goat-anti-mouse IgG1, anti-mouse IgG2a, or anti-mouse IgA (Southern Biotechnology Associates, Birmingham, AL), followed by the phosphatase substrate P-nitrophenyl phosphate (NPP; Sigma-Aldrich) in ethanolamine buffer (pH 9.8). Ab titers were defined on the linear portion of the curve as the interpolated dilution of a sample giving rise to an absorbance of 0.4 U above background.
In vitro stimulation of spleen cells
Spleen cells were obtained by mechanical dissociation and filtering through a nylon mesh. RBCs were subjected to lysis by osmotic shock using hypotonic Tris-ammonium chloride. After washing in HBSS (Life Technologies, Paisly, Scotland), the cells were resuspended in Iscove’s medium (Biochrom) supplemented with 10% heat-inactivated FCS, 50 µM 2-ME (Sigma-Aldrich), 1 mM L-glutamine (Biochrom), and 50 µg/ml gentamicin. Spleen cells (105 cells/well) were then cultured (8% CO2, 37°C) in round-bottom, 96-well microtiter plates (Nunc) in the presence or absence of varying concentrations of H. pylori lysate Ags or anti-CD3 by adding 10% supernatant from the 145-2Cll cell line (25). Cell-free supernatants were collected 96 h after incubation, and stored at -70°C until assayed for cytokine concentration.
Cytokine assays
The concentrations of IFN-, IL-4, IL-5, IL-10, or IL-13 in
the supernatants were assessed by ELISA. Briefly, 96-well round bottom
microtiter plates (Dynatech Laboratories, Chantilly, VA) were incubated
with 2.5 µg/ml rat anti-mouse IFN- (BD PharMingen) or 1–5
µg/ml anti-mouse IL-4 (Endogen, Woburn, MA), IL-5, or IL-10 (BD
PharMingen). The sample supernatants, or recombinant mouse IFN-,
IL-4, IL-5, or IL-10 (R&D Systems) standards were then added to the
appropriate wells. Bound IFN-, IL-4, IL-5, or IL-10 were detected by
sequential incubations with a polyclonal rabbit anti-IFN-
antiserum or biotinylated mAb to mouse IL-4 (Endogen), IL-5, or IL-10
(BD PharMingen), followed by AP-conjugated goat anti-rabbit Ig
(Southern Biotechnology Associated) for IFN-, or HRP-conjugated
anti-biotin (Vector Laboratories) for the IL-4, IL-5, and IL-10
assays. Finally, the AP substrate NPP in ethanolamine buffer (pH 9.8)
or the HRP substrate
2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid (ABTS;
Sigma-Aldrich) in 0.1 M citrate buffer (pH 4.5) was added to each well
and the extent of the reaction was read at 405 nm using a Titertek
multiscan spectrophotometer. The cytokine concentrations in stimulated
culture supernatants were estimated from the standard curves generated
with each cytokine. IL-13 was determined using a mouse-specific IL-13
ELISA kit according to the manufacturer’s instructions (R&D Systems).
The sensitivity of detection for the respective cytokine was 0.5 ng/ml
for IFN-, 40 pg/ml for IL-4, 0.05 ng/ml for IL-5, 0.5 ng/ml for
IL-10, and 1.5 pg/ml for IL-13.
ELISPOT
Single cell suspensions were prepared from the spleens of WT
mice immunized with lysate plus CT in the presence or absence of rIL-12
followed by challenge infection with H. pylori bacteria.
Ag-specific IFN--producing T cells were then determined by
ELISPOT assay as described by Gottwein et al.
(15).
Statistical analysis
Wilcoxon rank sum test was used for independent samples for analysis of significance in all experimental groups, except for the cytokine values which were compared by the one-tailed Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies in mice have shown that a primary
Helicobacter infection results in persistent colonization
and gastric inflammation associated with a predominantly Th1-type
response (12, 26, 27). Because IL-12 plays a central role
in Th1 development, and IFN- is an important Th1-effector molecule,
we investigated the susceptibility for H. pylori
colonization in the Th1-deficient IL-12-/- and
GKO mouse strains. We found that the level of colonization in
IL-12-/- and GKO mice was comparable to that of
C57BL/6 WT mice at 2 and 8 wk postinfection (Fig. 1
). At 2 wk postinfection, inflammatory
changes in IL-12-/-, GKO, and WT mice were
minimal, but at 8 wk more severe gastritis had developed in both
IL-12-/- and WT mice compared with that seen in
GKO mice (Table I). Thus, a primary
H. pylori infection could be established in the absence of
the proinflammatory cytokines IL-12 or IFN-, but it appeared that
IFN-, more than IL-12, was directly involved in the development of
chronic gastritis in the course of the infection.
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To investigate the protective role of Th1-type immune responses
against Helicobacter bacteria, we undertook studies in
Th1-defective, IL-12-/-, or GKO mice and
compared the results with those obtained in WT mice or
Th2low, IL-4-/- mice. The
mice were immunized orally with bacterial lysate plus CT adjuvant and
subsequently challenged with live H. pylori bacteria.
Whereas immunized Th1-deficient IL-12-/- and
GKO mice failed to show protection against infection, immunized
IL-4-/- and WT mice were significantly
(p < 0.01) protected (Fig. 2, A–B). Thus, while immune
protection exhibited a strict requirement for IL-12 and IFN-,
IL-4-dependent Th2-type responses appeared to be dispensable for the
development of protective immunity following mucosal vaccination. Of
particular note, the immunized and well-protected
IL-4-/- and WT strains both exhibited
significantly stronger infiltration of CD4+ T
cells in the gastric mucosa as compared with that observed in immunized
IL-12-/- and GKO mice (Fig. 2C).
Thus, protection was associated with an increased level of
CD4+ T cells in the gastric mucosa.
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). However, coadministration of rIL-12
increased IFN--producing splenic T cells by 5-fold (ELISPOT, not
shown) and further reduced the bacterial colonization by 4-fold
subsequent to a live challenge infection, clearly demonstrating the
importance of Th1-type responses for Helicobacter-specific
protective immunity (Table II). Delivery of rIL-12 to CT only or
PBS-treated mice had no statistically significant effect on reduction
of the bacterial burden upon challenge with live bacteria (Table II).
Also mice immunized with lysate alone with or without rIL-12 were
unprotected (not shown). Thus, not only does oral immunization,
as seen in the previous experiments, convey protection, but also
intranasal immunization is effective and can be further improved by the
addition of rIL-12 to the immunization regimen.
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Next, the relationship between protective immunity and the
development of gastritis under the influence of Th1 or Th2 functions
was examined. Protective mucosal immunization with lysate and CT
adjuvant resulted in pronounced gastritis in WT mice subsequent to a
challenge infection as compared with the gastric inflammatory response
observed in CT only treated control mice (Fig. 3) or unchallenged immunized mice (not
shown). Despite a similar level of protection, immunized
IL-4-/- mice displayed significantly lower
(p < 0.05) gastritis than WT mice upon
challenge with live bacteria (Fig. 3). Therefore, IL-4/Th2 cells may,
in fact, contribute to the severity of the gastritis seen after
immunization. Notwithstanding this, Th1 cells clearly played a central
role in the development of postimmunization gastritis, since the level
of inflammation was significantly attenuated in both the mutant strains
as compared with that in WT or IL-4-/- mice
(Fig. 3). In accordance with the gastritis score, the protected WT and
IL-4-/- mice also demonstrated substantial
alterations in epithelial cell morphology with hyperplasia counts of
1.19 ± 0.89 and 0.74 ± 0.7, respectively, in the corpus as
compared with that observed in the unprotected
IL-12-/- and GKO mice, which were 0.43 ±
0.63 and 0.53 ± 0.7, respectively. Orally immunized, but
unchallenged, mice exhibited no gastritis and had comparable
hyperplasia scores to those recorded in CT-immunized or naive control
mice (not shown).
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production by Th1
cells
We investigated the cytokine profiles of
Helicobacter-specific immune T cells derived from protected
or unprotected mice. Splenocytes were isolated and cultured in the
presence or absence of recall H. pylori lysate Ags. We found
that the protected and challenged IL-4-/- and
WT mice produced high levels of IFN- and good levels of IL-13 (Table III) but undetectable IL-4, IL-5, or
IL-10 (not shown). By contrast, unprotected and challenged
IL-12-/- and GKO mice failed to produce IFN-
or IL-4, IL-5, or IL-10 in response to recall Ag, but produced
significantly higher levels of IL-13 as compared with
IL-4-/- or WT mice (Table III). In particular,
GKO mice exhibited very strong IL-13 responses. Anti-CD3 stimulation of
splenic T cells confirmed the poor ability to produce IFN- in
IL-12-/- mice, whereas the IL-13 production was
lower than in IL-4-/- or WT mice and
substantially lower as compared with GKO mice. Thus, it appears that
protection against H. pylori infection in orally immunized
mice is independent of IL-4, IL-5, IL-10, and IL-13, but directly
related to IL-12 and up-regulated IFN- production by immune Th1
cells.
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Oral immunization in IL-12-/- mice with
lysate plus CT induced high levels of serum anti-H.
pylori IgG1, IgG2a, and IgA (Fig. 4A), which were comparable to
those observed in WT mice. As predicted immunized GKO mice exhibited
low levels of specific IgG2a Abs, whereas
IL-4-/- mice had strong specific IgG2a Ab
levels, but low levels of IgG1. Both the latter strains exhibited
reduced levels of specific serum IgA Abs as compared with
IL-12-/- or WT mice. Similar patterns of Ab
responses were also observed in gastric secretions of the immunized and
challenged mice, except that we observed more specific IgA Abs in
unprotected GKO mice as compared with protected
IL-4-/- or WT mice (Fig. 4B). The
specific Ab levels of the different isotypes in serum and gastric
secretions in immunized Th1- or Th2-deficient mice poorly reflected the
level of immune protection induced by oral immunization.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Furthermore,
coadministration of rIL-12 to intranasal immunizations in WT mice
substantially improved protection and reduced the bacterial load
subsequent to a live challenge infection. Contrary to the work of
others, we found that a Th2-type response did not play a major role in
protection, but interestingly appeared to contribute to the development
of the postimmunization gastritis, as IL-4-/-
mice had significantly less gastritis than WT mice (Fig. 3). Protection
was strongly associated with postimmunization gastritis and local
infiltration of CD4+ T cells in the gastric
mucosa. Only the well-protected strains,
IL-4-/- and WT mice, showed strong influx of
CD4+ T cells to the gastric mucosa. Splenic T
cells, isolated from the well-protected mice, demonstrated strong Th1
activity and, therefore, we assume that also Th1 cells dominated the
infiltrating CD4+ T cells in the gastric mucosa.
Although not formally proven, it is reasonable to believe that these
cells were responsible for reducing bacterial load and enhancing
gastric inflammation as both unprotected GKO and
IL-12-/- mice exhibited reduced T cell
infiltration and gastritis. Thus, immune protection involves a certain
level of gastric inflammation, but we do not believe that the
inflammation alone is mediating protection. Rather, postimmunization
gastritis is usually found in well-protected strains as a consequence
of Th1 cells invading the mucosa, but apart from inflammation, immune
protection also has other components in it as revealed in the
IL-4-/- mice, which exhibited less
postimmunization gastritis but were equally well-protected compared
with WT mice.
We used IFN-, IL-12, and IL-4 gene-disrupted mice to assess the
contribution of T cell subsets in protective immunity against H.
pylori infection. IL-12 has been shown to be important for IFN-
production by a variety of immune cells, including
CD4+ T cells, NK cells, activated B cells, and
macrophages (28, 29). IFN- production and delayed-type
hypersensitivity responses are markedly reduced in IL-12
p40-/- mice (18). A major function
of IL-12 is to promote the differentiation of Th1 cells (30, 31) and to serve as a costimulus for maximum IFN- secretion
by already committed Th1 cells (32). In the present study,
splenocytes from immunized and challenged
IL-12-/- mice produced no IFN- after
stimulation with recall Ag in vitro. This was not a result of poor
priming of CD4+ T cells in
IL-12-/- mice, as high levels of IL-13 were
observed in cultures with recall Ag. Moreover,
IL-12-/- mice exhibited comparable levels of
specific IgG1, IgG2a, and IgA to WT mice after oral immunization,
arguing against the idea that IL-12-/-
mice were poor responders to H. pylori Ags. However, IL-13
was not associated with protection as T cells from unprotected GKO mice
produced large amounts of IL-13 but were completely unprotected against
a live challenge infection.
Gastric colonization with H. pylori bacteria was not
influenced by the ability to respond with inflammation and a Th1
response, because at 2 and 8 wk after inoculation with live bacteria
all strains exhibited comparable levels of colonizing bacteria. These
findings are at variance with those of Sawai et al. (33),
who reported that a strain of H. pylori (KP48a) without the
ability to colonize C57BL/6 mice was able to colonize GKO mice,
suggesting that the ability to produce IFN- could impair H.
pylori colonization of the gastric epithelium. Contrary to these
findings, Eaton et al. (34) reported that H.
pylori colonization was not significantly different in SCID
recipients of IFN--deficient splenocytes than in recipients of
normal splenocytes, indicating that the absence of IFN- did not
affect the ability to colonize the gastric mucosa. In the present
study, we could not detect any influence of either IFN- or IL-12 on
the extent of primary colonization of the gastric mucosa. The GKO mice
exhibited less gastritis than IL-12-/- and WT
mice at 8 wk after infection, but were equally strongly colonized,
demonstrating that IFN- is important for the development of
gastritis.
The regulatory roles of Th1 and Th2 cells in immune protection against
H. pylori infection are incompletely understood. Only a few
reports have addressed the roles of Th subsets in
Helicobacter immunity and immunopathology (12, 13, 16). The study by Mohammadi et al. (12) argued
against a protective role of Th1 cells and IFN-, because in vivo
neutralization of IFN- had no effect on the level of colonizing
H. felis bacteria after challenge, but rather resulted in a
significant reduction of gastric inflammation. They also reported that
adoptive transfer of H. felis-specific Th2 cell lines
resulted in the reduction of H. felis colonization
(13). Thus, it was concluded that Th1 cells were more
responsible for the pathogenesis of Helicobacter infection
than protection, whereas the Th2 cells were involved in protection. By
contrast, the present data clearly demonstrates that Th1 cells and the
cytokines IFN- and IL-12 are crucial for the development of immune
protection against H. pylori infection. These factors are
also responsible for promoting the development of gastric inflammation.
Th2 cells, in contrast, as demonstrated by our findings in the
IL-4-/- mice, are not important for protection.
These mice developed significant protection comparable to that in WT
mice. However, Th2 cells, or IL-4 itself, may contribute to the
development of inflammation because the protected
IL-4-/- mice exhibited significantly less
postimmunization gastritis. Consistent with the notion that Th2 cells
are dispensable for protection, Aebischer et al. (16)
reported that IL-4R-/- mice (35, 36) were strongly protected against H. pylori
infection. Furthermore, adoptive transfer of
Helicobacter-specific spleen CD4+ T
cells into the IL-4R-/- mice induced
significant protection (37). Gottwein et al.
(15), in contrast, reported that both Th1 and Th2 cells
could induce protection. They found that systemic immunization with
lysate, together with either CFA or alum, resulted in
Helicobacter-specific immunity dominated by Th1 and Th2
cells, respectively, and both induced significant protection. In
support of the results of the present study, Jiang et al.
(17) showed that skewing toward a Th1-dominated
environment using a replication-defective adenovirus resulted in a
significant reduction of an established H. felis infection
in WT, but not in IFN- and IL-12-deficient, mice.
The mechanism(s) by which Th1 cytokines induce protective immunity or
reduce Helicobacter colonization is poorly understood. The
lack of IFN- and IL-12 may have some effect on the production of
specific Abs as, for instance, in GKO mice, IgG2a is reduced (Fig. 4)
(17). In contrast, Abs did not correlate with protection
in the present study and in previous studies using
IgA-/- and µMT mice significant protection
developed following immunization (9, 10). The
CD4+ T cells that were recruited into the gastric
mucosa of protected IL-4-/- and WT mice
affected the microenvironment by driving the development of gastritis,
and this could have direct implications for the ability to colonize the
epithelium. Postimmunization gastritis appears to be a consistent
finding in all models showing protection against
Helicobacter infection (8, 9, 23, 38, 39, 40).
Equally possible though, Th1 cells in the gastric mucosa could affect
other mechanisms that would affect bacterial survival. In this context,
the ability of macrophages to phagocytose and control bacterial
infection is under the strong influence of IFN- and IL-12 and may
play a role in the control also of gastric Helicobacter
infection. Moreover, IFN- could enhance APC functions of gastric
epithelial cells, thereby, focusing specific T cells to the epithelium
(41, 42). Such a mechanism coupled with an increased
apoptosis of epithelial cells, induced by the bacteria, could provide
unfavorable growth conditions for the bacteria to survive in the
gastric mucosa (43, 44, 45). However, the mild inflammation in
the protected IL-4-/- mice argues that also
other more direct effects of Helicobacter-specific gastric
CD4+ T cells may exist. It is not yet clear what
these effects could be and extended studies in the mouse model are,
therefore, much needed. Clearly, the immune response and gastritis seen
in the chronically infected mice and the postimmunization gastritis
appear to be very similar, but a fundamental difference is that the
latter hosts components that result in bacterial clearance. This was
recently demonstrated in an elegant study by Garhart et al.
(46), who found that immunized and protected mice
exhibited a transient bacterial colonization associated with gastritis,
but which subsides over time as the bacterial load diminishes. Thus,
prophylactic immunization does not prevent colonization by H.
pylori completely, but it adds a quality to the resistance against
infection that is not observed in unimmunized infected animals.
Understanding the fundamental difference between these two types of
responses is necessary for the development of effective prevention and
therapy against diseases caused by H. pylori infection.
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2 Address correspondence and reprint requests to Dr. Ali A. Akhiani, Department of Clinical Immunology, Göteborg University, Guldhedsgatan 10A, SE 413 46 Göteborg, Sweden. E-mail address: ali.akhiani{at}microbio.gu.se
3 Abbreviations used in this paper: CT, cholera toxin; WT, wild type.
Received for publication June 26, 2002. Accepted for publication October 4, 2002.
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),
GKO (
), and C57BL/6 WT (
) mice at 2 and 8 wk after inoculation
with live bacteria. Groups of 10 mice were infected with 3 x
108 CFU of H. pylori SS1 on two consecutive
days. The presence of H. pylori in gastric tissue was
assessed by quantitative culture (A) and urease activity
(B) as described in Materials and
Methods. The bars represent mean ± SEM of pooled data
from two to three experiments.
) and challenged with H. pylori bacteria. The
extent of histological gastritis was assessed 2 wk after challenge.
Data points are from individual mice. The bar represents the mean of
each group. Values of p given are by Wilcoxon rank sum
test. The mean scores of IL-12-/-, GKO, and
IL-4-/- mice immunized with Ly + CT were significantly
different from that in the immunized WT mice. We used both CT alone and
lysate alone (not shown) as negative controls. Similar results were
observed when we compared the Ly + CT group with Ly alone (not shown).
One representative experiment of three is shown.