Skip Navigation


International Immunology Advance Access originally published online on February 20, 2007
International Immunology 2007 19(4):435-446; doi:10.1093/intimm/dxm008
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
19/4/435    most recent
dxm008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (3)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Kiriya, K.
Right arrow Articles by Chiba, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kiriya, K.
Right arrow Articles by Chiba, T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Essential role of Peyer's patches in the development of Helicobacter-induced gastritis

Keiichi Kiriya, Norihiko Watanabe, Akiyoshi Nishio, Kazuichi Okazaki1, Masahiro Kido, Kazuyuki Saga, Junya Tanaka, Takuji Akamatsu, Shinya Ohashi, Masanori Asada, Toshiro Fukui and Tsutomu Chiba

Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
1 Present address: Department of Gastroenterology and Hepatology, Kansai Medical University, 2-3-1 Shinmachi, Hirakata, Osaka 573-1191, Japan

Correspondence to: T. Chiba; E-mail: chiba{at}kuhp.kyoto-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Helicobacter bacteria colonize in the stomach and induce strong, specific local and systemic humoral and cell-mediated immunity. Helicobacter binds to the host epithelial cells, directly triggering the recruitment of neutrophils. Local inflammatory processes in the gastric mucosa are followed by extensive immune cell infiltration, resulting in chronic active gastritis characterized by a marked infiltration of Th1 cytokine-producing CD4+ T cells. The mechanisms underlying the development of Th1 cell-mediated chronic gastritis, however, are not clear. Peyer's patches (PPs), the major inductive sites for mucosal immunity in the gut system, might orchestrate Helicobacter-specific local and systemic humoral and cell-mediated immunity. To examine the roles of PPs in the development of Helicobacter-induced gastritis, we generated PP-null mice that normally develop well-organized lymphoid organs except for PPs and intra-gastrically infected the resulting PP-null mice with Helicobacter felis. PP deficiency severely impaired both the development of Th1 cell-mediated gastritis induced by Helicobacter and the production of anti-Helicobacter antibodies despite marked bacterial colonization of the gastric mucosa. Although PP deficiency did not impair the differentiation of Helicobacter-specific CD4+ T cells into IFN-{gamma}—producing Th1 cells, Helicobacter-specific IFN-{gamma}—producing CD4+ T cells in PP-null mice lacked the ability to migrate into Helicobacter-colonized gastric mucosa. These findings suggest that PPs have an important role in Helicobacter-specific local and systemic humoral and cell-mediated immunity, including the development of Helicobacter-induced gastritis.

Keywords: CCR9 CD4+ T cells, IFN-{gamma}, production, migration, Peyer's patch-null mice


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Helicobacter bacteria colonize in the stomach and induce strong, specific local and systemic humoral and cell-mediated immunity (1, 2). Helicobacter does not invade epithelial cells of the gastric mucosa, but binds to the host epithelial cells (3, 4). Helicobacter activates signal transduction molecules in the epithelial cells and induces up-regulation of IL-8 expression, directly triggering the recruitment of neutrophils (5–8). The innate immunity mediated by neutrophils and macrophages is involved in the local inflammatory processes in the acute phase of gastritis, whereas the adaptive immunity mediated by infiltrating T cells and B cells has a major role in the chronic phase of gastritis and in specific humoral responses (1, 2).

Helicobacter-induced chronic gastritis, which triggers the development of peptic ulcer diseases, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma in humans (1, 2, 9), is characterized by a marked infiltration of CD4+ T cells, which produce large amounts of Th1 cytokines (10, 11). The mechanisms underlying the development of Th1 cell-mediated chronic gastritis induced by Helicobacter are not fully understood. Although the gastric mucosa originally does not have a lymphoid apparatus (12), Helicobacter-induced local inflammatory processes in innate immunity might trigger gastric T cell expansion and B cell differentiation. Because Helicobacter antigens can easily access the intestinal lumen distal to the stomach, however, it might be that these antigens induce mucosal immune responses in the well-organized MALT of the intestine.

Peyer's patches (PPs) are the major inductive sites for MALT in the gut system (13). The microfold cells (M cells) residing in the follicle-associated epithelium overlying PPs efficiently take up luminal antigens and micro-organisms. Resident PP–dendritic cells (DCs) process and present these molecules to T cells and orchestrate immune responses to luminal antigens and pathogens, including specific Ig production and Th1 cell-mediated immune responses (14-17). Therefore, PPs may have a role in inducing both the recruitment of the Helicobacter-specific Th1 cells into the Helicobacter-colonized gastric mucosa and the differentiation of anti-Helicobacter antibody-producing cells.

Because IL-7R{alpha} signal has an essential role in the embryonic formation of PPs, administration of anti-IL-7R{alpha} blocking mAb into C57BL/6 (B6) pregnant mice can transplacentally disturb PP formation in the offspring (18). The PP-null offspring normally develop well-organized lymphoid organs except for PPs (14, 18, 19). To examine the in vivo role of PPs in the development of Helicobacter-induced gastritis and in the production of anti-Helicobacter antibodies, we generated PP-null B6 mice by administering anti–IL-7R{alpha} mAb to B6 pregnant mice, then intra-gastrically infected the resulting PP-null mice with Helicobacter felis (H. felis). The development of Helicobacter-induced gastritis and the production of anti-Helicobacter antibodies were severely impaired in Helicobacter-infected PP-null mice, despite the marked colonization of bacteria in the gastric mucosa. These findings suggest that PPs have an essential role in the host immune response to Helicobacter infection.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
B6 were purchased from Japan SLC (Shizuoka, Japan) and mated in our animal facility. Female and male mice were mated overnight, and those with a vaginal plug were judged to be pregnant. Noon of the day when the vaginal plug was found was considered to be gestation day 0.5. We obtained manipulated PP-null B6 mice as described previously (18). In brief, timed-pregnant B6 mothers were intra-peritoneally injected with IL-7R{alpha} blocking mAb (2 mg; A7R34, a kind gift from S. Nishikawa, Kobe, Japan) on gestation day 14.5. All mouse protocols were approved by the Institute of Laboratory Animals at the Kyoto University Graduate School of Medicine.

Helicobacter felis and infection
Helicobacter felis is a gastric Helicobacter that colonizes in the stomach of laboratory mice, dogs and cats, and can induce chronic active gastritis (20–24). Helicobacter felis (ATCC49179) was purchased from the American Type Culture Collection (Rockville, MD, USA). The bacteria were grown in Brucella broth at a titer of 1 x 108 organisms per ml. The bacterial suspension was stored at –80°C until use. Normal B6 and PP-null mice (8 weeks old) were inoculated with 0.5 ml of bacterial suspension into the stomach using a steel catheter.

Bacterial staining
Helicobacter felis was labeled with the red fluorescent lipophilic dye PKH26, using a PKH26 red fluorescent cell linker mini kit (Sigma, St. Louis, MO, USA). Helicobacter felis was harvested from the culture medium, washed twice with PBS, adjusted to 4 x 108 ml–1 with labeling buffer and incubated with an equal volume of a PKH26 dilution (1:250 in labeling buffer) for 5 min at 25°C. The reaction was stopped with PBS containing 1% BSA. Stained bacteria were then washed three times with complete medium, and were finally resuspended in Brucella broth to a concentration of 1 x 108 ml–1. Fluorescence emission of bacteria was determined by fluorescence microscope before oral administration. Three days after inoculation of bacteria, mice were sacrificed, and the PPs and small intestines were immediately removed and were frozen for immunohistochemistry.

Immunohistologic analysis
Fluorescence immunohistology was performed on frozen sections as follows using FITC-conjugated anti-CD11c (HL3, BD Biosciences, San Jose, CA, USA), anti-CD4 (RM4-5, BD Biosciences) or anti-CD8 (53-6.7, eBioscience, San Diego, CA, USA). Sections of 6 µm were cut from tissue blocks and mounted onto glass slides. The sections were air-dried for 30 min, fixed in acetone for 5 min and blocked with PBS containing 10% non-fat dried milk for 30 min. The sections were stained with FITC-conjugated antibodies for 1 h. After the final wash, the slides were mounted by Vectashield (Vector Laboratories, Burlingame, CA, USA) and examined under a fluorescence microscope. In case of chemokine receptor staining, after using Avidin/Biotin Blocking kit (Vector Laboratories), the sections were stained with anti-CC chemokine receptor (CCR)-9 (242503, R&D Systems, Minneapolis, MN, USA) for 1 h, followed by staining using Vectastain Elite ABC Kit (Vector Laboratories) according to the manufacturer's instructions. And lastly the sections were incubated with Texas Red Avidin D (Vector Laboratories) or FITC-conjugated streptavidin (eBioscience) for 30 min.

Histologic examination
Mice were sacrificed 6–12 weeks after inoculation, and the stomachs, spleens, mesenteric lymph nodes (MLN), PPs and small intestines were immediately removed. Half of the stomach was collected for the assessment of H. felis colonization and histologic examination. Tissues were fixed in neutral buffered formalin, embedded in paraffin wax and cut into 4-µm thick sections. These sections were stained with hematoxylin and eosin for histopathology and May–Giemsa for the assessment of H. felis colonization. The other half of the stomach was frozen for immunohistochemistry. The degree of gastritis was determined according to the semi-quantitative scoring system as described previously (25). Chronic inflammation, characterized by the infiltration of mononuclear cells, was graded from 0 to 3, where 0 = no increase in the number of inflammatory cells, 1 = slight infiltration of the lamina propria (LP) by lymphocytes and plasma cells, 2 = moderately dense infiltration of the LP by lymphocytes and plasma cells and 3 = very dense lymphoplasma-cell infiltration in the LP. Activity, characterized by the presence of polymorphonuclear leukocytes, was graded from 0 to 3, where 0 = no increase in inflammatory cells, 1 = scattered neutrophils in the LP with no leukopedesis in the region of the gastric pits, 2 = moderate number of neutrophils in the LP with microabscesses in the region of the gastric pits and 3 = extensive neutrophils in the LP with obvious cryptitis. Atrophic changes were graded from 0 to 3 according to the loss of specialized cells, chief and parietal cells, (0 = no loss, 1 = mild loss of specialized cells, 2 = moderate loss of specialized cells and 3 = severe loss of specialized cells). The degree of colonization of H. felis in the infected gastric mucosa was assessed by the semi-quantitative scoring system as described previously (25). Bacterial colonization was graded from 0 to 4, where 0 = no bacteria, 1 = 1–2 bacteria per crypt, 2 = 3–10 bacteria per crypt, 3 = 11–20 bacteria per crypt, and 4 = >20 bacteria per crypt.

ELISA
An ELISA was used to measure serum levels of anti-H. felis antibody as described previously (26). Helicobacter felis antigens were extracted by sonication of bacteria in tris-buffered saline (pH 7.4, 0.01 mol l–1). Duplicate wells of microtiter plates (Nunc, Roskilde, Denmark) were incubated with antigens (100 µg ml–1) in PBS for 16 h at 4°C. The wells were blocked with PBS containing 2.5% non-fat dried milk and then incubated for 2 h at room temperature with serial dilutions of sera. The wells were then incubated with HRP-labeled goat anti-mouse IgG or goat anti-mouse IgA (Serotec, Oxford, UK) diluted at a predetermined concentration for 24 h at 4°C. After rigorous washing, each well was reacted with a substrate (o-phenylenediamine, Nacalai tesque, Kyoto, Japan) solution for 15 min. The reaction was terminated using 25 µl of 2 mol l–1 H2SO4, and optical denstiy (OD) was determined using a microplate reader set to 490 nm.

For quantitation of serum Ig isotype levels, serum samples were diluted to 1:24 000 for IgM, 1:120 000 for IgG and 1:6000 for IgA in PBS, were incubated in microtiter plates coated with antibodies to each isotype and then with alkaline phosphatase (AP)-labeled isotype-specific antibodies (Southern Biotechnology, Birmingham, AL, USA). ELISA color development was performed using phosphatase substrate p-nitrophenyl phospatate tablets (Sigma), and OD was determined using a microplate reader set to 405 nm.

Adoptive transfer
A total of 1.0 x 107 spleen cells from H. felis-infected or uninfected normal B6 and PP-null mice were injected intra-peritoneally three times every 4 days into H. felis-infected or uninfected RAG2–/– recipient mice. After 6 days after the completion of transfer, the mice were killed, and the spleen, small intestine and stomach were analyzed by immunohistologic staining with FITC-conjugated anti-CD4.

Flow cytometry
The following mAbs were used: FITC-conjugated anti-CD4 (RM4-5), biotinylated anti-CCR5 (C34-3448), allophycocyanin (APC)-conjugated anti-CD4 (GK1.5), anti-IFN-{gamma} (XMG1.2) and PE-conjugated anti-{alpha}4ß7 (DATK32) purchased from BD Biosciences and PE-conjugated anti-CCR6 (140706) and anti-CCR9 (242503) from R&D Systems. FITC-conjugated anti-CD8a (53-6.7) and PE–streptavidin was from eBioscience. For intracellular cytokine production, single cells were isolated from the spleen and cells were seeded at 4 x 106 per well in flat-bottomed 24-well plates in the presence of 4 x 106 ml–1 H. felis antigens. After 18 h of culture, cells were washed twice and re-stimulated with 50 ng ml–1 phorbol myristate acetate (PMA) (Sigma) + 2 µg ml–1 ionomycin (Sigma). After 3.5 h, brefeldin A (Sigma) was added at 10 µg ml–1. After 2.5 h, cells were collected, and stained for cell-surface molecules. Cells were fixed and permeabilized, using Fix & Perm Cell Permeabilization Kit (Caltag Laboratories, An Der Grub, Austria), and stained with APC-conjugated anti-IFN-{gamma}. Flow cytometric analysis was performed as described previously (27).

Real-time quantitative reverse transcription–PCR
The gastric tissues of B6 mice before and 1 day, 2 weeks, 4 weeks, and 12 weeks after H. felis infection were used for real-time quantitative reverse transcription (RT)–PCR. Total RNA was extracted with the Qiagen RNeasy mini kit (Qiagen). RT was done with SuperScript RT II (Invitrogen, Carlsbad, CA, USA). The real-time quantitative reactions were performed with ABI Prism 7300 detection system (Applied Biosystems, Foster City, CA, USA) according to manufacture's instructions. The following primers were used: thymus-expressed chemokine (TECK): 5'-CCGGCATGCTAGGAATTATCA-3' and 5'- GGCACTCCTCACGCTTGTACT-3' and glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5'-CAACTTTGTCAAGCTCATTTCC-3' and 5'-GGTCCAGGGTTTCTTACTCC-3'. Values are expressed as arbitrary units (relative to GAPDH x105).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Helicobacter can be taken up by PPs in association with CD11c+ cells in PPs
Because PPs are the major inductive sites for MALT in the gut system and efficiently take up luminal antigens and micro-organisms, we first examined whether Helicobacter can be taken up by PPs more efficiently than by the other sites of the small intestine. Helicobacter felis was labeled by a red fluorescent dye PKH26 and PKH-conjugated H. felis was intra-gastrically injected into B6 mice. Immunohistologic analyses using frozen sections of the small intestine 3 days after the injection showed that PKH-H. felis antigens were mainly located in the dome regions of PPs comparing with other sites of the small intestine (Fig. 1 and data not shown). Visualized H. felis antigens were localized in the CD11c+ cells (Fig. 1). These results suggest that Helicobacter can be taken up by PPs more efficiently than the other sites of the small intestine and that CD11c+ cells in PPs take up H. felis antigens and may induce Helicobacter-specific antigen priming.


Figure 1
View larger version (52K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. Immunohistologic findings of PPs in normal B6 mice after inoculation of PKH-conjugated H. felis. Helicobacter felis was labeled by a red fluorescent dye PKH26 and PKH-conjugated H. felis was intra-gastrically inoculated into B6 mice (A). After 3 days after the inoculation, the small intestine were isolated and stained with FITC-conjugated anti-CD11c antibody. PKH-conjugated H. felis antigens (red) were mainly located in the dome regions of PPs (arrow head, C and I). Visualized H. felis antigens were localized in the CD11c+ cells (B–D and H–J). (E–G) and (K)–(M) are shown controls of PPs in the uninoculated mice. (H)–(M) are shown in the dome regions of PPs. Original magnification: x100–400 as shown in the panels.

 
PP-null mice develop well-organized lymphoid organs except for PPs
Helicobacter infection induces chronic gastritis that is characterized by a marked infiltration of CD4+ T cells and the infiltrating CD4+ T cells have a major role in the chronic phase of gastritis (10, 11). In addition, Helicobacter infection induces B cell activation and specific humoral responses (1, 2). We first examined whether uninfected PP-null mice develop well-organized lymphoid organs except for PPs. In uninfected PP-null mice, the thymus normally developed and the absolute numbers of CD4+, CD8+ T cells and B cells in the spleen, MLN and inguinal lymph nodes (ILN) were comparable with those in B6 mice (Fig. 2A and B, and data not shown). The peritoneal cavity contained a comparable number of the lymphocytes and the percentages of B cell subsets (data not shown). In addition, the serum levels of IgM, IgG and IgA in uninfected PP-null mice were similar to those in B6 mice (Fig. 2C). These data suggest that cellular composition of the remaining lymphoid organs and general humoral responses are normal in PP-null mice as described previously (14, 18, 19).


Figure 2
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. Cellular composition of the lymphoid organs and general humoral responses in uninfected normal B6 and PP-null mice. (A) We isolated cells from the spleen (SPL), MLN, ILN and PP of uninfected normal B6 (C57BL/6 wild type; WT) and PP-null mice (PP null), and stained cells with APC-conjugated anti-CD4 and FITC-conjugated anti-CD8 antibodies. Percentages of CD4+ and CD8+ T cells in the total viable cells are shown. (B) Numbers of CD4+ and CD8+ T cells in each lymphoid organs were calculated by (percentage of indicated cells in viable cells) x (number of viable cells). The data are representative of three mice ND, not detected (C) The serum levels of total IgM, IgG and IgA determined by ELISA. Bars indicate the mean of each group and horizontal short bars indicate the standard deviation. Student's unpaired t-test was used to compare the values between two groups. NS, not significant,, P > 0.05.

 
Development of Helicobacter-induced gastritis was impaired in Helicobacter-infected PP-null mice
Because B6 mice are sensitive to H. felis infection (21), infected B6 mice develop chronic active gastritis 12 weeks after H. felis infection, which mimics the pathologic features observed in Helicobacter pylori-induced gastritis in humans (21, 26). To examine the in vivo role of PPs in the development of Helicobacter-induced gastritis, we intra-gastrically infected PP-null and B6 mice with H. felis. Twelve weeks after H. felis infection, macroscopic examination revealed that the gastric mucosa was thicker in H. felis-infected B6 mice, but not in H. felis-infected PP-null mice (data not shown). Histologic examination revealed that the gastric mucosa in H. felis-infected B6 mice had chronic gastritis with severe lymphocyte infiltration, loss of parietal and chief cells and hyperplasia of the mucus neck cells (Fig. 3A). These findings were further confirmed by a gastritis scoring system that evaluates (i) chronic inflammation, characterized by the infiltration of mononuclear cells; (ii) activity, characterized by the presence of polymorphonuclear leukocytes and (iii) atrophic changes based on the loss of parietal and chief cells (Fig. 3B). In contrast, H. felis-infected PP-null mice showed limited inflammation of the gastric mucosa without any glandular atrophy or foveolar hyperplasia (Fig. 3A and B). These data suggest that PP-null mice do not develop Helicobacter-induced chronic active gastritis.


Figure 3
View larger version (89K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. Histologic findings of the gastric mucosa and gastritis score of H. felis-infected normal B6 and PP-null mice. (A) H. felis-infected B6 mice (C57BL/6 WT) developed chronic active gastritis 12 weeks after infection. H. felis-induced gastritis was characterized by a marked infiltration of lymphocytes, glandular atrophy, and mucosal hyperplasia (right upper panel). In contrast, H. felis-infected PP-null mice (PP null) showed no inflammation of the gastric mucosa, and no glandular atrophy or foveolar hyperplasia (right lower panel). Original magnification: x100. (B) The degree of gastritis was determined according to the semi-quantitative scoring system, as described in Methods. Closed bars indicate the mean of each group; horizontal short bars indicate the standard error. Student's t-test for unpaired data was used to compare the values between two groups. Asterisks indicate P < 0.05.

 
Anti-Helicobacter antibody production was impaired in Helicobacter-infected PP-null mice
To examine whether PP deficiency influences the production of anti-Helicobacter antibodies, we used an ELISA to examine the serum levels of anti-H. felis antiobodies in normal B6 mice and PP-null mice 12 weeks after H. felis infection. The increases in serum anti-H. felis IgG and IgA levels in H. felis-infected PP-null mice were significantly smaller than those in H. felis-infected B6 mice (Fig. 4A). In addition, although anti-H. felis antibody titers were proportional to the gastritis scores in H. felis-infected B6 mice, there was no such tendency in H. felis-infected PP-null mice (Fig. 4B). These results suggest that PP deficiency impairs not only the development of Helicobacter-induced gastritis but also the production of anti-Helicobacter antibodies. Although previous studies showed that antibody production can reduce the severity of the gastric inflammation in Helicobacter infection (28, 29), our results suggest that the impairment of the development of H. felis-induced gastritis is not due to excessive production of pathogen-specific antibodies in PP-null mice.


Figure 4
View larger version (42K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. The serum levels of anti-H. felis IgG and IgA and H. felis gastric colonization in H. felis-infected normal B6 and PP-null mice. (A) Serum levels of anti-H. felis antibodies in normal B6 (C57BL/6 WT) and PP-null mice (PP null) with (closed bar) or without (open bar) H. felis infection. Anti-H. felis IgG is shown in the left panel and IgA in the right panel. We used ELISA to examine anti-H. felis antibodies 12 weeks after H. felis infection. Bars indicate the mean of each group and horizontal short bars indicate the standard error. Student's unpaired t-test was used to compare the values between two groups. Asterisks indicate P < 0.05. (B) Relationship between serum levels of anti-H. felis antibodies and gastritis score in H. felis-infected normal B6 (open circle) and PP-null mice (closed circle). The degree of gastritis was assessed as described in the Methods. (C) Helicobacter felis-infected PP-null mice showed marked colonization of H. felis in the gastric mucosa (arrow head). Original magnification: x400. (D) Helicobacter felis colonization score in H. felis-infected PP-null mice (PP null) was significantly higher than that in H. felis-infected normal B6 mice (C57BL/6 WT). The degree of H. felis colonization in the infected gastric mucosa was assessed as described in the Methods. Bars indicate the mean of each group. Student's unpaired t-test was used to compare the values between two groups. Asterisk indicates P < 0.05.

 
Helicobacter felis markedly colonized in the gastric mucosa of H. felis-infected PP-null mice
To evaluate the possibility that H. felis infection in PP-null mice was quickly resolved, and did not result in chronic inflammation of the gastric mucosa, we examined the degree of H. felis colonization in H. felis-infected PP-null mice. Although H. felis-infected PP-null mice did not develop gastritis, H. felis was extensively colonized in the gastric mucosa of these mice and the colonization scores in PP-null mice were significantly higher than those in normal B6 mice (Fig. 4C and D), excluding the possibility of a colonization defect or a quick resolution for H. felis infection in H. felis-infected PP-null mice.

PP deficiency did not impair the differentiation of Helicobacter felis-specific T cells into Th1 cells
Helicobacter-induced chronic gastritis is characterized by a marked infiltration of IFN-{gamma}—producing CD4+ T cells. To investigate whether the differentiation of H. felis-specific T cells into Th1 cells is impaired in H. felis-infected PP-null mice, we isolated spleen cells from normal B6 and PP-null mice 6 weeks after H. felis infection and cultured them in the presence of H. felis antigens followed by stimulation with PMA and ionomycin. IFN-{gamma} production was assessed by intracellular cytokine staining followed by flow cytometry. Helicobacter felis-specific re-stimulation induced a increase in IFN-{gamma}—producing CD4+ T cells in the spleen cells of H. felis-infected B6 mice as compared with those in uninfected B6 mice (data not shown). Importantly, re-stimulation of H. felis antigens also induced IFN-{gamma}—producing CD4+ T cells in the spleen cells of H. felis-infected PP-null mice. After H. felis antigen re-stimulation, the percentage of IFN-{gamma}—producing cells in CD4+ T cells of H. felis-infected PP-null mice (0.83 ± 0.38) was not significantly lower than that of H. felis-infected B6 mice (0.96 ± 0.49). These results suggest that PP deficiency does not impair the differentiation of H. felis-specific T cells into Th1 cells.

PP deficiency did not alter the migration of gut-homing T cells to the LP of the small intestine, but severely impaired Helicobacter felis-specific T cell infiltration into the stomach
PP–DCs direct immune responses to luminal antigens and pathogens, determining the migration of CD4+ T cells into MALT in the gut system (15). Therefore, we performed immunohistologic examination to assess the migration capacity of non-specific conventional gut-homing CD4+ T cells into the small intestine and that of H. felis-specific CD4+ T cells into the gastric mucosa after H. felis infection.

Before and after H. felis infection, the LP of the small intestine contained a comparable number of CD4+ T cells in both B6 and PP-null mice, suggesting that PP deficiency does not alter the migration capacity of non-specific conventional gut-homing CD4+ T cells in the physiological MALT system (Fig. 5A–F). In contrast to the small intestine, because of the absence of physiological MALT system in the gastric mucosa, gastric mucosa of both normal B6 mice and PP-null mice do not have any CD4+ T cells before H. felis infection (Fig. 5G and J). After H. felis infection, infiltrating lymphocytes in the gastric mucosa of B6 mice mainly consisted of CD4+ T cells, but not CD8+ T cells, whereas neither CD4+ T cells nor CD8+ T cells were detected in the gastric mucosa of PP-null mice (Fig. 5H, I, K and L and data not shown), suggesting that the bacteria colonization in the stomach induces the infiltration of CD4+ T cells in normal B6 mice, but not in PP-null mice. These data suggest that PP deficiency may not alter physiological migration of gut-homing T cells to the LP of small intestine, but may impair the infiltration of H. felis-specific CD4+ T cells into the H. felis-colonized gastric mucosa.


Figure 5
View larger version (79K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. Immunohistologic findings of small intestinal and gastric mucosa of uninfected and H. felis-infected normal B6 and PP-null mice. (A–F) The LP of small intestine contained a comparable number of CD4+ T cells in both normal B6 (WT) and PP-null mice regardless of the presence of the H. felis infection. (G–L) In contrast, because of the absence of physiological MALT system in the gastric mucosa, gastric mucosa of both normal B6 and PP-null mice do not have any CD4+ T cells before H. felis infection (G and J). After H. felis infection, infiltrating lymphocytes in the gastric mucosa of H. felis-infected B6 normal mice (H and I) mainly consisted of CD4+ T cells, whereas few CD4+ T cells were detected in the gastric mucosa of H. felis-infected PP-null mice (K and L). HE, hematoxylin and eosin staining of the same views shown with anti-CD4 antibody staining. Original magnification: x100.

 
To test this possibility further, we transferred spleen cells from PP-null and B6 mice into the peritoneal cavity of RAG2–/– mice and analyzed lymphoid tissues 6 days after the completion of transfer. When we transferred spleen cells from uninfected PP-null and B6 mice into uninfected RAG2–/– mice, a large number of CD4+ T cells could be detected in the LP of small intestine but not in the gastric mucosa of recipient uninfected mice regardless of the presence of the PP in donor mice (Fig. 6A, B, G and H). Next, when we used H. felis-infected PP-null and B6 mice for donor mice, CD4+ T cells could be detected only in the LP of small intestine but not in the gastric mucosa of uninfected recipient mice (Fig. 6C, D, I and J). Taken together, the migration of CD4+ T cells into the LP of small intestine in these settings appears to represent the constitutive recruitment of conventional CD4+ T cells and uninfected gastric mucosa is not the site for the constitutive recruitment of conventional CD4+ T cells. Importantly, uninfected gastric mucosa do not allow the infiltration of bacteria-specific CD4+ T cells, suggesting that the infiltration of bacteria-specific CD4+ T cells depends on H. felis colonization in the gastric mucosa. Last, H. felis-colonized gastric mucosa of the recipient RAG2–/– mice allows the infiltration of CD4+ T cells of H. felis-infected B6 donor mice but not those from the H. felis-infected PP-null donor mice (Fig. 6E, F, K and L), suggesting that PP deficiency severely impairs the infiltration of H. felis-specific CD4+ T cells into the H. felis-colonized gastric mucosa.


Figure 6
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6. Transfer of spleen cells from PP-null and B6 mice into RAG2–/– mice in uninfected or H. felis-infected condition. Spleen cells from PP-null and B6 mice were injected into the peritoneal cavity of RAG2–/– mice in the condition of H. felis infection shown in panels. After 6 days after the completion of transfer, the small intestine and stomach were isolated and stained with FITC-conjugated anti-CD4 antibody. When we transferred spleen cells from uninfected or H. felis-infected B6 (WT) and PP-null mice into uninfected RAG2–/– mice, a large number of CD4+ T cells could be detected in the LP of small intestine but not in the gastric mucosa of recipient uninfected mice regardless of the presence of the PP in donor mice (A–D and G–J). In contrast to the LP of small intestine, H. felis-colonized gastric mucosa of the recipient RAG2–/– mice allows the infiltration of CD4+ T cells of H. felis-infected B6 donor mice but not those from the H. felis-infected PP-null donor mice (E, F, K and L). Original magnification: x200.

 
Helicobacter felis-specific Th1 cytokine-producing cells in PP-null mice lacked the ability to migrate into H. felis-colonized gastric mucosa
PP–DCs instruct naive T cells to differentiate into gut-homing T cells that express gut–homing chemokine receptors, such as integrin {alpha}4ß7, and CCR9 (15) and infiltrating T cells in Helicobacter-induced gastritis express these chemokine receptors (30–32). CCR5 is a chemokine receptor expressed on Th1 cells that promotes their migration and CCR5–CCL5 interaction is indispensable for the induction of acute graft–versus–host disease mediated by PPs (14). CCR6 is expressed on CD4+ T cells and CCR6—MIP-3{alpha}/CCL20 interaction play an important role in mucosal immunity (33, 34) Therefore, we firstly analyzed CD4+ T cells in PPs and the spleen of uninfected normal B6 and PP-null mice for the anti-chemokine receptor antibodies. Freshly isolated total CD4+ T cells in PPs of uninfected B6 mice contained the large populations expressing CCR5, CCR6 and CCR9 but not integrin {alpha}4ß7 (Fig. 7A, upper panels). However, the percentage of chemokine receptor-expressing cells in CD4+ T cells of the spleen in uninfected PP-nulll mice was not lower than those in B6 mice (Fig. 7A, middle and lower panels). The relative size of the CD4+ T cell compartments expressing chemokine receptors are directly proportional to the CD4+ T cell number because total CD4+ T cell numbers in the spleen did not vary between uninfected PP-null and B6 mice (Fig. 2A and B).


Figure 7
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7. Chemokine receptor expression of CD4+ T cells. (A) Chemokine receptor expression of CD4+ T cells in uninfected mice. We isolated PP and spleen (SPL) cells from normal B6 (C57BL/6 WT) and spleen cells from PP-null mice (PP null), and stained cells with anti-chemokine receptor antibodies for CCR5, CCR6, CCR9 and integrin {alpha}4ß7. Data shown are phenotypes of CD4+ T cells. The numbers in histograms show percentages of chemokine receptor-expressing cells. (B) Chemokine receptor expression of IFN-{gamma}—producing CD4+ T cells in H. felis-infected mice. We isolated spleen cells from normal B6 and PP-null mice at 9 weeks after H. felis infection and cultured them under stimulation with or without H. felis antigens followed by stimulation with PMA and ionomycin. We examined expression levels of chemokine receptors, CCR5, CCR6 and CCR9 of IFN-{gamma}—producing CD4+ T cells. Anti-IFN-{gamma} mAb was used for intracellular cytokine staining. Cell-surface markers and intracellular IFN-{gamma} expression were determined by flow cytometry. Data shown are phenotypes of IFN-{gamma}—producing CD4+ T cells. The numbers in histograms show percentages of chemokine receptor-expressing cells. Data represent one of five experiments.

 
To further examine the roles of chemokine receptor expression of H. felis-specific IFN-{gamma}—producing CD4+ T cells in H. felis-infected mice, we isolated spleen cells from H. felis-infected normal B6 mice and PP-null mice at 9 weeks after H. felis infection and re-stimulated these cells with or without H. felis antigens, followed by the stimulation of PMA and ionomycin. Without H. felis antigen re-stimulation ex vivo, non-specific IFN-{gamma}—producing CD4+ T cell populations that express chemokine receptors, CCR5, CCR6 and CCR9 were comparable between H. felis-infected normal B6 mice and PP-null mice (Fig. 7B, left panels). In contrast, after H. felis antigen re-stimulation, the percentage of IFN-{gamma}—producing CD4+ T cell population expressing CCR9, but not CCR5 and CCR6 in H. felis-infected PP-null mice, was markedly lower that of H. felis-infected normal B6 mice (Fig. 7B, right panels). These data suggest that PP deficiency impairs differentiation of H. felis-specific CD4+ T cells into CCR9-expressing cells that have the capacity to migrate into the gastric mucosa.

We then investigated expression of CCR9 and CCR9 ligand, TECK/CCL25 in H. felis-induced inflamed gastric mucosa. Immunohistologic examination of the gastric mucosa revealed that CCR9-expressing cells could be detected in the inflamed gastric mucosa of H. felis-infected B6 mice, but not in that of uninfected B6 mice (Fig. 8A and data not shown). CCR9-expressing cells in the LP of gastric mucosa (Fig. 8B, upper panels) and in the submucosa of the stomach (Fig. 8B, middle panels) are CD4 positive, whereas non-gut-homing CD4+ T cell population in the ILN do not have any CCR9-expressing cells (Fig. 8B, lower panels). In addition, real-time quantitative RT–PCR analysis revealed that H. felis infection can significantly induce the expression of CCR9 ligand, TECK/CCL25 in the stomach of B6 mice (Fig. 8C). Taken together, TECK/CCL25–CCR9 interaction in gastric mucosa may be involved in the migration of H. felis-specific CD4+ T cells into the H. felis-infected gastric mucosa.


Figure 8
View larger version (37K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8. CCR9 and TECK/CCL25 expression in H. felis-infected gastric mucosa. (A) CCR9-positive cells (green) were detected in gastric mucosa of H. felis-infected B6 mice. Original magnification: x200. (B) CCR9-expressing cells (red) in the LP of gastric mucosa (a–c) and in the submucosa of the stomach (d–f) are CD4 positive (green), whereas non-gut-homing CD4+ T cell population in the ILN (g–i) do not have any CCR9-expressing cells. Original magnification: x200 (a–c) and x400 (d–i). (C) Total RNA was extracted from the gastric tissues of B6 mice before and 1 day, 2, 4 and 12 weeks after H. felis infection. Using the real-time quantitative RT–PCR analysis, relative TECK mRNA level is evaluated. The means and standard deviation of each group are indicated. Student's t-test for unpaired data was used to compare with the value of day 0. Asterisks indicate P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In the present study, we generated PP-null mice by administering IL-7R{alpha} blocking mAb into the dams during pregnancy (18). PP-null mice developed well-organized lymphoid organs except for PPs, and general systemic humoral immunity was normal in PP-null mice. These findings are consistent with previous reports describing PP-null mice (14, 18, 19). Thus, PP-null mice are good models for studying the pathophysiologic roles of PPs in various gastrointestinal diseases. Interestingly, in H. felis-infected PP-null mice, both the development of gastritis and H. felis-specific antibody production were severely disturbed despite marked H. felis colonization in the gastric mucosa. These data clearly demonstrated that PPs have critical roles in host immune responses to Helicobacter infection. Importantly, in PP-null mice, the differentiation of H. felis-specific CD4+ T cells into CCR9-expressing cells was disturbed. Thus, failure of both the development of H. felis-induced gastritis and the production of H. felis-specific antibodies in PP-null mice might be due to the loss of migration ability of H. felis-specific CD4+ T cells into the gastric mucosa. In support of such an idea, previous studies demonstrated that Helicobacter and its secreted products enhance the expression of CCR9 on activated T cells and integrin {alpha}4ß7 is indispensable for the migration of CD4+ T cells to the gastric mucosa in Helicobacter infection (30–32). Feng et al. (35) reported that during rotavirus infection, IgA plasmablast migration into the small intestine requires integrin {alpha}4ß7 along with either CCR9 or CCR10. In addition, Staton et al. (36) showed that direct migration of CD8+ recent thymic emigrants into the small intestine requires CCR9–CCL25 interaction and integrin {alpha}4ß7. Therefore, integrin {alpha}4ß7 in addition to CCR9 may be required for the infiltration of H. felis-specific CD4+ T cells into the bacteria-colonized gastric mucosa.

Helicobacter-induced chronic gastritis is characterized by a marked infiltration of IFN-{gamma}—producing CD4+ T cells (10, 11). Moreover, the development of Helicobacter-induced gastritis is severely impaired in mice lacking CD4+ T cells or IFN-{gamma} production (37, 38). These data indicate that IFN-{gamma}—producing CD4+ T cells are essential for the development of chronic gastritis by Helicobacter. The present study demonstrated that in H. felis-infected PP-null mice, there was no infiltration of CD4+ T cells in the gastric mucosa, although there were IFN-{gamma}—producing CD4+ T cells specific for H. felis among the spleen cells. Together with previous reports, the present findings suggest that PPs have a critical role in instructing Helicobacter-specific IFN-{gamma}—producing CD4+ T cells to migrate into the gastric mucosa.

Recent studies indicate that both PP–DCs and MLN DCs derived from PP and the LP of small intestine can instruct T cells to express gut-homing chemokine receptors, integrin {alpha}4ß7, and CCR9, and to migrate into gut mucosal tissues (15, 39). In this study, we showed that CD4+ T cells in PP-null mice maintained their migration capacity into the LP of the small intestine, whereas Helicobacter-specific CD4+ T cells in PP-null mice did not migrate into Helicobacter-colonized gastric mucosa. These results suggest that PP–DCs and/or MLN DCs derived from PP but not MLN DCs derived from the LP of small intestine are involved in the instruction of Helicobacter-specific CD4+ T cells to differentiate into the gastric mucosa-homing Th1 cells.

Previous studies suggested that immune cells in PPs and LP of the small intestine have distinct roles in gastrointestinal immune responses. For instance, donor CD8+ T cell infiltration into host PPs is essential for Th1-type responses in acute graft–versus–host diseases (14). In contrast, the induction of oral tolerance for soluble antigens does not depend on PPs (40, 41). In gastrointestinal infection, such as those by Salmonella typhimurium, Toxoplasma gondii and rotavirus, PP T cells produce IFN-{gamma} (42–45). Efficiency of Th1-type protective immune responses against Eimeria vermiformis, a gut-residing apicomplexan parasite depends on the presence of PPs (46). We demonstrated here that the induction of Th1 cell-mediated gastritis in Helicobacter infection is also dependent on PPs. Therefore, PPs might be essential sites for the induction of Th1 immune responses in the gastrointestinal infection by non-invasive type organism.

The finding that the production of anti-Helicobacter antibodies was severely impaired in PP-null mice indicates that PPs have a critical role in the systemic production of anti-Helicobacter antibodies. Previous studies have reported that mucosal Ig responses to oral immunization for soluble antigens do not require PPs (40, 41, 47). Therefore, taken together with our data, it is possible that PP-dependent and PP-independent mucosal Ig responses exist in the MALT system and that they have distinct roles in the gut mucosal immune responses to luminal soluble antigens and pathogens.

Helicobacter bacteria orally infect and then bind to epithelial cells of the gastric mucosa by multiple surface adhesion molecules and subsequently activate signal transduction molecules in the epithelial cells (3–7). These direct interactions between Helicobacter and epithelial cells trigger the recruitment of neutrophils, resulting in acute inflammation of the gastric mucosa (1, 2, 8). Inflammation of the gastric mucosa, however, persists and induces chronic gastritis in virtually all Helicobacter-infected humans and some of laboratory animals (1, 2, 21). We demonstrated here that PP-null mice did not develop Helicobacter-induced chronic active gastritis despite marked bacterial colonization in the gastric mucosa. These findings might suggest that a local inflammatory process within the gastric mucosa directly induced by Helicobacter is dispensable for development of Helicobacter-induced chronic gastritis, whereas PPs in MALT of the intestine have an essential role in the development of chronic gastritis.

In conclusion, we demonstrated the indispensable roles of PPs in the host immune responses to Helicobacter infection, including the development of chronic gastritis. Although PPs are the most well-recognized secondary lymphoid organs in the intestine in both humans and mice, their distribution in the small intestine is different between humans and mice. PPs in mice are located on the antimesenteric border along the entire length of the small intestine, whereas in humans, PP structures are primarily clustered in the ileum (48, 49). Thus, further studies are required to confirm that PPs in humans also have critical roles in determining the host immune response to Helicobacter infection.


   Acknowledgements
 
We thank D. Wylie for assistance in preparation of the manuscript. This work is supported by Grants-in-aid for Scientific Research, 17590634, 18012029 18015028, 18209027 and 18590679 from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grant-in-Aid for Research on Measures for Intractable Diseases and Research on Advanced Medical Technology from the Ministry of Health, Labor and Welfare, Japan and Grant-in-Aid by Takeda Science Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research and The Shimizu Foundation for the Promotion of Immunology Research.


   Abbreviations
 
APC, allophycocyanin
B6, C57BL/6
CCR, CC chemokine receptor
DC, dendritic cell
H. felis, Helicobacter felis
ILN, inguinal lymph node
LP, lamina propria
MALT, mucosa-associated lymphoid tissue
MLN, mesenteric lymph node
OD, optical density
PP, Peyer's patch
PMA, phorbol myristate acetate
RT, reverse transcription

   Notes
 
Transmitting editor: M. Miyasaka

Received 17 June 2006, accepted 18 January 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Suerbaum S and Michetti P. (2002) Helicobacter pylori infection. N. Engl. J. Med. 347:1175.[Free Full Text]
  2. Farinha P and Gascoyne RD. (2005) Helicobacter pylori and MALT lymphoma. Gastroenterology 128:1579.[CrossRef][Web of Science][Medline]
  3. Guruge JL, Falk PG, Lorenz RG, et al. (1998) Epithelial attachment alters the outcome of Helicobacter pylori infection. Proc. Natl Acad. Sci. USA 95:3925.[Abstract/Free Full Text]
  4. Ilver D, Arnqvist A, Ogren J, et al. (1998) Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by retagging. Science 279:373.[Abstract/Free Full Text]
  5. Crabtree JE, Farmery SM, Lindley IJ, Figura N, Peichl P, Tompkins DS. (1994) CagA/cytotoxic strains of Helicobacter pylori and interleukin-8 in gastric epithelial cell lines. J. Clin. Pathol. 47:945.[Abstract/Free Full Text]
  6. Crabtree JE, Covacci A, Farmery SM, et al. (1995) Helicobacter pylori induced interleukin-8 expression in gastric epithelial cells is associated with CagA positive phenotype. J. Clin. Pathol. 48:41.[Abstract/Free Full Text]
  7. Covacci A and Rappuoli R. (2000) Tyrosine-phosphorylated bacterial proteins: Trojan horses for the host cell. J. Exp. Med. 191:587.[Free Full Text]
  8. Innocenti M, Thoreson AC, Ferrero RL, et al. (2002) Helicobacter pylori-induced activation of human endothelial cells. Infect. Immun. 70:4581.[Abstract/Free Full Text]
  9. Chiba T, Seno H, Marusawa H, Wakatsuki Y, Okazaki K. (2006) Host factors are important in determining clinical outcomes of Helicobacter pylori infection. J. Gastroenterol. 41:1.[CrossRef][Web of Science][Medline]
  10. Itoh T, Wakatsuki Y, Yoshida M, et al. (1999) The vast majority of gastric T cells are polarized to produce T helper 1 type cytokines upon antigenic stimulation despite the absence of Helicobacter pylori infection. J. Gastroenterol. 34:560.[CrossRef][Web of Science][Medline]
  11. Harris PR, Smythies LE, Smith PD, Dubois A. (2000) Inflammatory cytokine mRNA expression during early and persistent Helicobacter pylori infection in nonhuman primates. J. Infect. Dis. 181:783.[CrossRef][Web of Science][Medline]
  12. Genta RM, Hamner HW, Graham DY. (1993) Gastric lymphoid follicles in Helicobacter pylori infection: frequency, distribution, and response to triple therapy. Hum. Pathol. 24:577.[CrossRef][Web of Science][Medline]
  13. Newberry RD and Lorenz RG. (2005) Organizing a mucosal defense. Immunol. Rev. 206:6.[CrossRef][Web of Science][Medline]
  14. Murai M, Yoneyama H, Ezaki T, et al. (2003) Peyer's patch is the essential site in initiating murine acute and lethal graft-versus-host reaction. Nat. Immunol. 4:154.[CrossRef][Web of Science][Medline]
  15. Mora JR, Bono MR, Manjunath N, et al. (2003) Selective imprinting of gut-homing T cells by Peyer's patch dendritic cells. Nature 424:88.[CrossRef][Medline]
  16. Johansson C and Kelsall BL. (2005) Phenotype and function of intestinal dendritic cells. Semin. Immunol. 17:284.[CrossRef][Web of Science][Medline]
  17. Sato A and Iwasaki A. (2005) Peyer's patch dendritic cells as regulators of mucosal adaptive immunity. Cell. Mol. Life Sci. 62:1333.[CrossRef][Web of Science][Medline]
  18. Yoshida H, Honda K, Shinkura R, et al. (1999) IL-7 receptor {alpha}+ CD3- cells in the embryonic intestine induces the organizing center of Peyer's patches. Int. Immunol. 11:643.[Abstract/Free Full Text]
  19. Hamada H, Hiroi T, Nishiyama Y, et al. (2002) Identification of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine. J. Immunol. 168:57.[Abstract/Free Full Text]
  20. Dick E, Lee A, Watson G, O'Rourke J. (1989) Use of the mouse for the isolation and investigation of stomach-associated, spiral-helical shaped bacteria from man and other animals. J. Med. Microbiol. 29:55.[Abstract/Free Full Text]
  21. Lee A, Fox JG, Otto G, Murphy J. (1990) A small animal model of human Helicobacter pylori active chronic gastritis. Gastroenterology 99:1315.[Web of Science][Medline]
  22. Lee A, Krakowka S, Fox JG, Otto G, Eaton KA, Murphy JC. (1992) Role of Helicobacter felis in chronic canine gastritis. Vet. Pathol. 29:487.[Abstract]
  23. Fox JG, Blanco M, Murphy JC, et al. (1993) Local and systemic immune responses in murine Helicobacter felis active chronic gastritis. Infect. Immun. 61:2309.[Abstract/Free Full Text]
  24. Otto G, Hazell SH, Fox JG, et al. (1994) Animal and public health implications of gastric colonization of cats by Helicobacter-like organisms. J. Clin. Microbiol. 32:1043.[Abstract/Free Full Text]
  25. Sakagami T, Dixon M, O'Rourke J, et al. (1996) Atrophic gastric changes in both Helicobacter felis and Helicobacter pylori infected mice are host dependent and separate from antral gastritis. Gut 39:639.[Abstract/Free Full Text]
  26. Ohana M, Okazaki K, Oshima C, et al. (2001) A critical role for IL-7R signaling in the development of Helicobacter felis-induced gastritis in mice. Gastroenterology 121:329.[CrossRef][Web of Science][Medline]
  27. Watanabe N, Hanabuchi S, Soumelis V, et al. (2004) Human thymic stromal lymphopoietin promotes dendritic cell-mediated CD4+ T cell homeostatic expansion. Nat. Immunol. 5:426.[CrossRef][Web of Science][Medline]
  28. Akhiani AA, Schon K, Franzen LE, Pappo J, Lycke NY. (2004) Helicobacter pylori-specific antibodies impair the development of gastritis, facilitate bacterial colonization, and counteract resistance against infection. J. Immunol. 172:5024.[Abstract/Free Full Text]
  29. Akhiani AA, Stensson A, Schon K, Lycke NY. (2005) IgA antibodies impair resistance against Helicobacter pylori infection: studies on immune evasion in IL-10-deficient mice. J. Immunol. 174:8144.[Abstract/Free Full Text]
  30. Michetti M, Kelly CP, Kraehenbuhl JP, Bouzourene H, Michetti P. (2000) Gastric mucosal {alpha}4ß7-integrin-positive CD4 T lymphocytes and immune protection against Helicobacter infection in mice. Gastroenterology 119:109.[CrossRef][Web of Science][Medline]
  31. Quiding-Jarbrink M, Ahlstedt I, Lindholm C, Johansson EL, Lonroth H. (2001) Homing commitment of lymphocytes activated in the human gastric and intestinal mucosa. Gut 49:519.[Abstract/Free Full Text]
  32. Hatanaka K, Hokari R, Matsuzaki K, et al. (2002) Increased expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and lymphocyte recruitment in murine gastritis induced by Helicobacter pylori. Clin. Exp. Immunol. 130:183.[CrossRef][Web of Science][Medline]
  33. Varona R, Zaballos A, Gutierrez J, et al. (1998) Molecular cloning, functional characterization and mRNA expression analysis of the murine chemokine receptor CCR6 and its specific ligand MIP-3alpha. FEBS Lett. 440:188.[CrossRef][Web of Science][Medline]
  34. Varona R, Villares R, Carramolino L, et al. (2001) CCR6-deficient mice have impaired leukocyte homeostasis and altered contact hypersensitivity and delayed-type hypersensitivity responses. J. Clin. Invest. 107:37.[CrossRef]
  35. Feng N and Jaimes MC, et al. (2006) Redundant role of chemokines CCL25/TECK and CCL28/MEC in IgA plasmablast recruitment to the intestinal lamina propria after rotavirus infection. J. Immunol. 176:5749.[Abstract/Free Full Text]
  36. Staton TL and Habtezion A, et al. (2006) CD8+ recent thymic emigrants home to and efficiently repopulate the small intestine epithelium. Nat. Immunol. 7:482.[CrossRef][Web of Science][Medline]
  37. Smythies LE, Waites KB, Lindsey JR, Harris PR, Ghiara P, Smith PD. (2000) Helicobacter pylori-induced mucosal inflammation is Th1 mediated and exacerbated in IL-4, but not IFN-{gamma}, gene-deficient mice. J. Immunol. 165:1022.[Abstract/Free Full Text]
  38. Eaton KA, Mefford M, Thevenot T. (2001) The role of T cell subsets and cytokines in the pathogenesis of Helicobacter pylori gastritis in mice. J. Immunol. 166:7456.[Abstract/Free Full Text]
  39. Johansson-Lindbom B, Svensson M, Pabst O, et al. (2005) Functional specialization of gut CD103+ dendritic cells in the regulation of tissue-selective T cell homing. J. Exp. Med. 202:1063.[Abstract/Free Full Text]
  40. Fujihashi K, Kato H, van Ginkel FW, et al. (2001) A revisit of mucosal IgA immunity and oral tolerance. Acta. Odontol. Scand 59:301.[CrossRef][Web of Science][Medline]
  41. Kraus TA, Brimnes J, Muong C, et al. (2005) Induction of mucosal tolerance in Peyer's patch-deficient, ligated small bowel loops. J. Clin. Invest. 115:2234.[CrossRef][Web of Science][Medline]
  42. George A. (1996) Generation of gamma interferon responses in murine Peyer's patches following oral immunization. Infect. Immun. 64:4606.[Abstract]
  43. Karem KL, Kanangat S, Rouse BT. (1996) Cytokine expression in the gut associated lymphoid tissue after oral administration of attenuated Salmonella vaccine strains. Vaccine 14:1495.[CrossRef][Web of Science][Medline]
  44. Liesenfeld O, Kosek JC, Suzuki Y. (1997) Gamma interferon induces Fas-dependent apoptosis of Peyer's patch T cells in mice following peroral infection with Toxoplasma gondii. Infect. Immun. 65:4682.[Abstract]
  45. VanCott JL, Franco MA, Greenberg HB, et al. (2000) Protective immunity to rotavirus shedding in the absence of interleukin-6: Th1 cells and immunoglobulin A develop normally. J.Virol. 74:5250.[Abstract/Free Full Text]
  46. Kwa SF, Beverley P, Smith AL. (2006) Peyer's patches are required for the induction of rapid Th1 responses in the gut and mesenteric lymph nodes during an enteric infection. J. Immunol. 176:7533.[Abstract/Free Full Text]
  47. Yamamoto M, Rennert P, McGhee JR, et al. (2000) Alternate mucosal immune system: organized Peyer's patches are not required for IgA responses in the gastrointestinal tract. J. Immunol. 164:5184.[Abstract/Free Full Text]
  48. Lorenz RG and Newberry RD. (2004) Isolated lymphoid follicles can function as sites for induction of mucosal immune responses. Ann. N. Y. Acad. Sci. 1029:44.[CrossRef][Web of Science][Medline]
  49. Cornes JS. (1965) Peyer's patches in the human gut. Proc. R. Soc. Med. 58:716.[Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Immunol.Home page
A. Sayi, E. Kohler, I. Hitzler, I. Arnold, R. Schwendener, H. Rehrauer, and A. Muller
The CD4+ T Cell-Mediated IFN-{gamma} Response to Helicobacter Infection Is Essential for Clearance and Determines Gastric Cancer Risk
J. Immunol., June 1, 2009; 182(11): 7085 - 7101.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
T. Hashizume, A. Togawa, T. Nochi, O. Igarashi, M.-N. Kweon, H. Kiyono, and M. Yamamoto
Peyer's Patches Are Required for Intestinal Immunoglobulin A Responses to Salmonella spp.
Infect. Immun., March 1, 2008; 76(3): 927 - 934.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
19/4/435    most recent
dxm008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (3)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Kiriya, K.
Right arrow Articles by Chiba, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kiriya, K.
Right arrow Articles by Chiba, T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?