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The Journal of Immunology, 2003, 171: 4359-4368.
Copyright © 2003 by The American Association of Immunologists

Th1-Biased Tertiary Lymphoid Tissue Supported by CXC Chemokine Ligand 13-Producing Stromal Network in Chronic Lesions of Autoimmune Gastritis 1

Tomoya Katakai*, Takahiro Hara*, Manabu Sugai*, Hiroyuki Gonda*,{dagger} and Akira Shimizu2,*,{dagger}

* Center for Molecular Biology and Genetics, Kyoto University, and {dagger} Translational Research Center, Kyoto University Hospital, Kyoto, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Secondary lymphoid tissue is developmentally programmed and characterized by well-ordered compartmentalization of lymphocyte subsets and specialized stromal cells supporting the tissue architecture. By contrast, tertiary lymphoid tissue is defined as that induced in ectopic sites by inflammation, although its immunological role is largely unknown. In this study, we characterize the lymphoid tissue induced in the chronic lesion of murine autoimmune gastritis (AIG). Within the lymphoid cluster in the gastric mucosa, there is a clear segregation of T and B cells. Follicle-like B cell areas are always located on the luminal side of the mucosa, while T cells are located in the basal part. A typical lymphoid reticular network and follicular dendritic cells support the structure. Importantly, complement receptor 1+ follicular dendritic cells within the follicle express a B cell homing chemokine, CXC chemokine ligand 13. The number and size of the clusters correlate with the age of the mice and the serum autoantibody titer, suggesting the functional importance of the clusters in local Ab production, although involvement of the autoantibody in the disease progression is still unclear. AIG gastric lesions are known to constitute a Th1-biased, memory T cell-dependent immunomicroenvironment. The expression pattern of cytokines, including lymphotoxin-{beta}, and chemokines in the AIG stomach is consistent with this observation. Taken together, these facts suggest that, during the chronic phase of autoimmunity, long-lasting lymphocyte infiltration probably induces a unique tertiary lymphoid tissue that has a function distinct from that of regional lymph nodes. These neolymphoid tissues may maintain the local self reactivity supporting the vicious cycle of Th1-type reaction as well as autoantibody production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chronic inflammation is a kind of long-lasting immune reaction against pathogens, tumors, or self components, characterized by an accumulation of lymphocytes within local tissue. This situation is almost always accompanied by the functional destruction of the tissue. Infiltrating lymphocytes are likely to originate from secondary lymphoid tissue, such as draining lymph nodes, which have been activated in an Ag-specific or bystander fashion (1). A continuous Ag supply from the target tissue may be a driving force in maintaining the chronic phase of immune reactions. Especially in autoimmune diseases, self components released by the destructive inflammation within the target organ may further stimulate specific effector cells and trigger a vicious cycle. However, it is largely unknown whether the extravasated lymphocyte subsets are randomly located or following some rules within the tissue. Whether the infiltrating lymphocytes stay in the lesion for a long period or are replaced quickly is also unclear. There may be recirculation of lymphocytes between regional lymph nodes and the inflamed tissue. It has been reported that lymphoid structures resembling secondary lymphoid tissue develop at the inflamed site in several diseases (2, 3, 4, 5, 6, 7), and such ectopically induced lymphoid tissue was defined as tertiary lymphoid tissue (8).

In this study, we found organized lymphoid clusters in the chronic lesions in a murine experimental model for autoimmune gastritis (AIG). 3 The location of T cells, B cells, reticular networks (RN), and follicular dendritic cells (FDCs) in the cluster was analyzed by multicolor immunohistochemistry and confocal microscopy. Cytokine and chemokine expression in gastric mucosa (GM) and its relationship to autoantibody production in AIG were also assessed. These analyses revealed a unique feature of the lymphoid cluster, indicating that it is similar to, but clearly different from secondary lymphoid tissue in several respects, including the absence of typical germinal centers (GCs), high endothelial cell markers, naive T cells, Th2 cells, and Th2-type factors. We conclude that this gastric lymphoid tissue is a novel type of mucosal lymphoid tissue developed in the chronically Th1-biased autoimmune environment in the stomach of AIG.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

BALB/c mice were purchased from Japan SLC (Shizuoka, Japan). The mice were maintained at the facility in Center for Molecular Biology and Genetics, Kyoto University. AIG was induced by neonatal thymectomy of BALB/c mice 3 days after birth and diagnosed by ELISA for the detection of autoantibody in sera, as described previously (9, 10, 11). Six to thirty-two weeks after thymectomy, mice suffering from AIG were used for experiments.

Immunohistochemistry

Glandular stomachs and lymph nodes were isolated from the mice, embedded in OTC compound (Sakura Finetechnical, Tokyo, Japan), and frozen using liquid nitrogen. Cryosections (10–20 µm) were fixed with cold acetone for 5 min, treated with 0.05% Tween 20-PBS containing 1% BSA and 5% mouse serum, and stained with the following Abs: biotin anti-H+/K+-ATPase (1H9) (12), biotin anti-CD3, FITC anti-B220, anti-complement receptor 1 (CR1), anti-peripheral node adressin (PNAd), anti-syndecan-1 (BD PharMingen, San Diego, CA), anti-platelet endothelial cell adhesion molecule-1 (PECAM-1; Caltag, Burlingame, CA), anti-ER-TR7 (Serotec, Oxford, U.K.), anti-Mac-1 (Immunotech, Luminy, France), biotin anti-CXC chemokine ligand 13 (CXCL13), biotin anti-CC chemokine ligand 21 (CCL21) (R&D Systems, Minneapolis, MN), FITC anti-mouse IgM, PE anti-mouse IgD (Southern Biotechnology, Birmingham, AL), or FITC anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). PE anti-rat IgG Ab (Caltag), biotin anti-mouse IgM Ab (BD PharMingen), PE streptavidin, and APC streptavidin (Molecular Probes, Eugene, OR) were used as the secondary reagents. GC B cells were detected with FITC peanut agglutinin (PNA; Sigma-Aldrich, St, Louis, MO). After staining, sections were mounted using PermaFluor (Shandon, Pittsburgh, PA) and examined by confocal laser-scanning microscopy (MRC-1024; Bio-Rad, Osaka, Japan). The digital images obtained were analyzed using Adobe Photoshop software (Adobe Systems, San Jose, CA). To produce a view of the whole lymph node, images at different positions were assembled into a single image by combining adjacent images on using this software.

Preparation of GM-infiltrating cells and flow cytometric analysis

Mononuclear cells infiltrating into the GM were collected, as described previously (10, 11). In brief, 10% FCS-RPMI medium was injected into the submucosa of the stomach, which were then cut into small pieces to release infiltrating cells. After removing large tissue debris by passing through cotton in a funnel, the cells were used for flow cytometry. Isolated cells were stained with FITC anti-Thy-1.2, PE anti-CD4, FITC anti-CD8 (BD Biosciences, Mountain View, CA), biotin anti-B220 (Caltag), FITC anti-CD5, FITC anti-Mac-1, or FITC anti-B220 (BD PharMingen) Ab, and then analyzed on a FACSCalibur flow cytometer using CellQuest software (BD Biosciences).

RT-PCR

Semiquantitative RT-PCR analysis was performed, as described previously, with some modifications (11, 13). In brief, total RNA was prepared using TRIzol (Life Technologies, Gaithersburg, MD). Oligo(dT)12–18-primed cDNA was synthesized from 2 µg of RNA using Superscript II reverse transcriptase (Life Technologies). Five-fold serial dilutions of cDNA were analyzed by PCR in 10-µl reactions with ExTaq DNA polymerase (Takara, Otsu, Japan) and the following specific primer pairs: IFN-{gamma}, 5'-TGAACGCTACACACTGCATCTTGG-3' and 5'-CGACTCCTTTTCCGCTTCCTGAG-3'; IL-4, 5'-ATGGGTCTCAACCCCCAGCTAGT-3' and 5'-GCTCTTTAGGCTTTCCAGGAAGTC-3'; lymphotoxin-{beta} (LT-{beta}), 5'-CATCTTGCCCTCACCCTCTA-3' and 5'-AAACGCTTCTTCTTGGCTCG-3'; CCL4 (macrophage-inflammatory protein-1{beta}), 5'-CAGCTCTGTGCAAACCTAAC-3' and 5'-TCAGTTCAACTCCAAGTCAC-3'; CCL5 (RANTES), 5'-TCTGAGACAGCACATGCATC-3' and 5'-CCTAGCTCATCTCCAAATAG-3'; CXCL9 (monokine induced by IFN-{gamma}), 5'-CCAACACAGTGACTCAATAG-3' and 5'-TTATGTAGTCTTCCTTGAACG-3'; CXCL10 (IFN-inducible protein-10), 5'-AGACATCCCGAGCCAACCTT-3' and 5'-GTTAAGGAGCCCTTTTAGAC-3'; CCL17 (thymus- and activation-regulated chemokine), 5'-TCTGCTTCTGGGGACTTTTC-3' and 5'-GTTCGCCTGTAGTGCATAAG-3'; CCL22 (macrophage-derived chemokine), 5'-CTGGTCATTAGACACCTGAC-3' and 5'-CCCTAGGACAGTTTCTGGAG-3'; CCL19 (Epstein-Barr virus-induced molecule 1 ligand chemokine), 5'-GCACACAGTCTCTCAGGCTC-3' and 5'- CTCTCTTCTGGTCCTTGGTT-3'; CCL21 (secondary lymphoid tissue chemokine (SLC)), 5'-AGCTATGTGCAAACCCTGAG-3' and 5'-TCATAGGTGCAAGGACAAGG-3'; CXCL13 (B-lymphocyte chemoattractant), 5'-TTGAACTCCACCTCCAGGCA-3' and 5'-CTTCAGGCAGCTCTTCTCTT-3'; CXCL12 (stromal-derived factor-1), 5'-AAACCAGTCAGCCTGAGC TAC-3' and 5'-TTACTTGTTTAAAGCTTTCTC-3'; GAPDH, 5'-CCATCACCATCTTCCAGGAG-3' and 5'-CCTGCTTCACCACCTTCTTG-3'. Values were standardized relative to that for GAPDH as an internal control. The number of PCR cycles was as follows: 32 (IFN-{gamma}), 30 (IL-4), 30 (LT-{beta}), 30 (CCL4), 27 (CCL5), 28 (CXCL9), 28 (CXCL10), 30 (CCL17), 30 (CCL22), 30 (CCL19), 30 (CCL21), 30 (CXCL13), 29 (CXCL12), and 21 (GAPDH).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AIG develops in BALB/c mice subjected to thymectomy at 3 days after the birth (d3-Tx), and is an experimental model for the CD4+ T cell-mediated autoimmune disorder induced at high incidence (~50–80%) in mice that undergo d3-Tx (9, 10, 11). Disease-bearing mice (AIG mice) exhibit a characteristic profile of chronic inflammation in the stomach, with features such as the thickening of the GM due to epithelial hyperplasia (Fig. 1, A and B), selective loss of parietal cells (Fig. 1C), lymphocyte infiltration into the lamina propria (Fig. 1B), and the production of autoantibodies against parietal cells (12) (data not shown). In addition, the gastric lymph node (GLN) located at the lesser curvature is also enlarged (Fig. 1A). Notably, we have observed that the infiltrating lymphocytes frequently form clearly isolated clusters that are located at the basal part of the lamina propria adjacent to the muscularis mucosae, and are frequently associated with blood vessels (Fig. 1B, arrowheads; Fig. 2A, arrows).



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  		FIGURE 1. Histopathology of AIG in BALB/c mice. A, Gross photographs of dissected stomach and associated regional lymph node (GLN, arrow) from normal (left) and AIG (right) mice. The lower image shows the luminal surface view of the glandular stomach. B and C, Sections of GM from normal (left) and AIG (right) mice stained with hematoxylin (B), or anti-H+/K+-ATPase Ab detecting the location of parietal cells (C), respectively. Arrowheads indicate the lymphocytic infiltration forming a large lesional lymphoid cluster.

 


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  		FIGURE 2. Typical tissue architecture of the lymphoid clusters in the GM lesions. Serial frozen sections of stomach isolated from AIG mice were stained with Abs against CD3 (T cells), B220 (B cells), PECAM-1 (endothelial cells), ER-TR7 (reticular fibroblast), CR1 (follicular dendritic cells), or Mac-1 (myeloid cells), and analyzed by confocal imaging. A, Transverse view of GM containing three isolated lymphoid clusters (arrows). B, Serial transverse sections of a cluster. Clear T/B segregation, associated blood vessel (arrowhead), and lymphoid RN (arrow) are shown. C, Three independent clusters in apical-basal plane sections.

 
Lymphoid tissue-like structure of the lymphoid clusters

Inside secondary lymphoid tissue such as lymph nodes and the spleen, T and B lymphocytes are each located in separate regions known as the T cell area of the paracortex (detected with anti-CD3 Ab) and the B cell follicle (detected with anti-B220 Ab), respectively (Fig. 3A). Two types of stromal cells of mesenchymal origin support these structures. A RN constructed of fibroblastic reticular cells covers a large part of the lymph node with a complex meshwork that is stained with ER-TR7 Ab (Fig. 3B), while follicles and GCs are backed by FDC networks that can be detected with anti-CR1 (CD35) Ab (Fig. 3C). In contrast to those from normal animals, GLNs from AIG mice become greatly hypertrophic, with dramatically expanded RN, and develop many GCs, reflecting extensive lymphocyte activation, especially of B cells.



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  		FIGURE 3. Tissue architecture of GLN as assessed by confocal fluorescence immunohistochemistry. Serial frozen sections of GLN isolated from normal and AIG mice were stained with several Abs to detect CD3 (T cells), B220 (B cells) (A), ER-TR7 (reticular fibroblasts) (B), or CR1 (follicular dendritic cells) (C). All images are shown at the same magnification.

 
To determine whether any lymphoid structure is organized at these focal lesions or not, we performed confocal fluorescence immunohistochemical analysis of the sections of GM from AIG mice (Fig. 2). To our surprise, a large fraction of lymphocytic clusters (>100 µm), but not of relatively scattered infiltrates, had clear segregation of the T and B cells. Each cluster had a single follicle-like B cell aggregate and associated T cell area. It is worth noting that the B cell follicle within a lymphoid cluster was always located on the luminal side of the mucosa, while the T cells were positioned at the relatively basal part. The characteristic lymphoid RN was distributed precisely at the cluster. Moreover, we detected an FDC marker, CR1, at the B cell area, indicating that a follicular structure like that in secondary lymphoid tissue had indeed developed in these clusters. Mac-1+ myeloid cells were distributed around the clusters (Fig. 2C). No typical lymphocytic infiltration or lymphoid structure was observed in normal mouse GM (Fig. 1B).

Development of mature B cell follicles requires a homeostatic chemokine, CXCL13 (B-lymphocyte chemoattractant), expressed by stromal cells (14, 15). Gene targeting has revealed the critical importance of this chemokine for the organization of normal follicular architecture (14). Therefore, we next assessed whether CXCL13 was expressed in the B cell area of the clusters in GM lesions (Fig. 4, A and B). As a positive control experiment, anti-CXCL13 polyclonal Ab was shown to stain a part of the CR1+ FDC network in a thin, filamentous pattern in the follicles of the GLNs from AIG mice. Control goat Ab showed some background staining, but no structural pattern. Strikingly, the CXCL13 expression could also clearly be detected on the CR1+ FDC-like network on the follicles of GM lymphoid clusters. However, some of these clusters exhibited very weak or undetectable signals, probably due to CXCL13 expression levels below the limit of experimental sensitivity (data not shown). Despite a formation of obvious follicular structure, typical GCs (as detected by PNA) were rarely observed even in the largest lymphoid clusters (Fig. 5A).



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  		FIGURE 4. CXCL13 expression in FDCs at the GM follicle. A, Serial frozen sections of GLNs and GM from AIG mice were stained with several combinations of Abs and analyzed by confocal imaging. B, CXCL13+ FDC network on B cell follicle in GM (upper panels) and higher magnification view focused on the cluster stained with CR1, CXCR13, or ER-TR7 (lower panels). C, Schematic representation of a typical GM lymphoid tissue. B, B cell area (follicle); T, T cell area; FRC, fibroblastic reticular cell; BV, blood vessel; EP, epithelial cell layer; LP, lamina propria; MM, muscularis mucosae; PC, plasma cell; SM, submucosa.

 


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  		FIGURE 5. A, Absence of typical GC morphology in GM follicle. Serial frozen sections of GLNs and GMs from AIG mice were stained with FITC-conjugated PNA and Abs against several markers and analyzed by confocal imaging. Mature GCs were easily detected at follicles in GLNs (arrow). B, Absence of PNAd and CCL21 (SLC) expression on endothelial cells associated with GM tertiary lymphoid tissue. Serial sections were stained with Abs against endothelial cell marker PECAM-1 or HEV marker PNAd or SLC. Asterisks indicate the positions of GM follicles on the serial sections. No remarkable signals for PNAd or CCL21 were detected in the GM lymphoid tissue (arrows). Arrowheads show some nonspecific staining of PNAd on gastric epithelial cells. C, Extrafollicular distribution of IgG+syndecan-1+ plasma cells in the GM lesions (arrow). Serial sections were stained with Abs against Ig subclasses and a plasma cell marker, syndecan-1.

 
We previously showed that almost all T cells infiltrating into the GM of AIG mice are memory cells (11). Homing of naive T cells to LNs is primarily mediated by the expression of PNAd, carbohydrate ligands for L-selectin, and a chemokine, CCL21 (SLC), on high endothelial venules (HEV) (16, 17, 18). HEVs in the GLN from AIG mice expressed these molecules strongly (Fig. 5B), and naive T cells are present in the GLN to some extent (11). In contrast, we detected no substantial signals for PNAd or SLC in the GM lymphoid tissues, suggesting that the absence of these factors at the lesional endothelial cells prevents the migration of naive T cells into the GM.

Taken together, these findings demonstrate that the architecture of the lymphoid clusters developed in the GM is well organized and embodies some aspects of secondary lymphoid tissue. We conclude that these clusters are kind of tertiary lymphoid tissues induced by chronic inflammation. Because the animals used in this study were maintained under conventional conditions, some infectious agent(s) might have influenced the development of lymphoid tissue in the AIG animals. However, even under specific pathogen-free conditions, we have observed obviously clustered lymphocytic infiltration in the GM lesion (data not shown), and the clusters had the same features of tertiary lymphoid tissue as shown above.

Number and size of GM lymphoid tissue, and correlation with autoantibody titer in sera

The number of lymphoid clusters observed in the sections along the greater curvature of the stomach wall in AIG mice increased gradually as the age of the mice increased (Table I). In addition, the size of each cluster was larger (data not shown) and the maturation of the FDC network as determined by CR1 or CXCL13 staining was more advanced in older AIG mice than in younger ones (Table I). These findings support the idea that the long-term retention of activated lymphocytes in peripheral tissue enables the development of organized lymphoid architecture. We also found that the number of the clusters and the titer of anti-GM autoantibody in the serum were significantly correlated (Fig. 6). Therefore, the lymphoid cluster development in the GM lesion should be a valuable hallmark for evaluating AIG progression. These findings also suggest that tertiary lymphoid tissues in the target organ play a pivotal role in the autoantibody production by self-specific B cells and in disease progression, although the definitive evidences are as yet absent.


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  		Table I. Summary of tertiary lymphoid tissue development in the GM of AIG mice

 


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  		FIGURE 6. Correlation between the number of tertiary lymphoid tissue and the serum autoantibody titer. Titers of serum anti-GM autoantibodies were determined by ELISA as the greatest dilution at which the absorbance at 450 nm was >0.1. Lymphoid tissue lesion numbers were determined by counting the CD3+/B220+ clusters observed in the sections along the greater curvature of the glandular stomach wall from the cardia to the pylorus. Data from various animals (age 6–24 wk) were plotted on a semilogarithmic.

 
Immune cell subsets in the GM of AIG mice

To assess the composition of the immune cell subsets infiltrating into chronic GM lesions, we checked several markers on isolated cells and compared the GM, GLNs, and PBLs from normal and AIG mice by flow cytometry (Fig. 7). As expected, AIG mice suffered from T cell lymphopenia caused by the neonatal thymectomy, resulting in a marked decrease in the cells positive for T cell markers such as Thy-1.2, CD4, and CD8 in blood as well as in the GLN. By contrast, B cells were markedly increased, especially in the GLNs of AIG mice, reflecting the vigorous proliferation of B cells in addition to T lymphopenia. Interestingly, the cell population in the inflamed GM exhibited some similarity to that in normal lymph nodes as to T cell-B cell ratio, although the CD4+ and CD8+ composition was slightly different and Mac-1+ cells constituted a substantial fraction of the cells within the GM of AIG mice. A striking difference was observed between the cell populations in the GM and the GLN in AIG mice, suggesting that the ongoing immune reactions in these two sites are quite different. We could not isolate enough leukocytes from the GM of normal mice to analyze the cell populations (see Fig. 8, normal GM profiles).



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  		FIGURE 7. Composition of immune cell subsets in PBL, GLN, and GM from normal and AIG mice. Isolated cells from more than five mice (age 10–20 wk) were stained with various Abs and analyzed by flow cytometry. The results are shown as the percentage of positive cells in the leukocyte gate.

 


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  		FIGURE 8. Infiltration of conventional B cells, but not B-1 cells, into the GM lesions of AIG mice. Isolated cells stained with B220/CD5 or B220/Mac-1 were analyzed by flow cytometry. Results are shown as dot plots. B-1 cells (B220+CD5+, B220+Mac-1+) and B-2 cells (B220+CD5-, B220+Mac-1-) were gated, respectively, and shown as the percentage in leukocyte gated cells.

 
It seemed possible that peritoneal B-1 cells migrate to the GM and form the follicle-like B cells area of the lymphoid clusters, because B-1 cells are highly sensitive to CXCL13 attraction (19, 20, 21). We checked this point by staining GM cells with B-1 markers (Fig. 8). Typical B-1 cells in the peritoneal cavity showed the B220+CD5+Mac-1+ phenotype in normal controls, while a large part of the B220+ cells in the GLN were CD5-Mac-1- conventional B (B-2) cells, and this was also the case in AIG mice. In the GM of AIG mice, most B220+ cells expressed neither CD5 nor Mac-1, thus indicating that the follicular structure in the GM is composed of conventional B cells derived from the circulation. B cells within the GM follicle were actually IgM+IgD+ follicular-type B cells comparable to those in secondary lymphoid organs (Fig. 5C). In contrast, IgG+syndecan-1+B220low plasma cells were distributed outside the follicles and scattered beneath the gastric epithelial layer (Fig. 5C, arrows). This situation again corresponds to that in the secondary organ in which plasma cells are located in extrafollicular sites, especially in the medula or red pulp (22).

Cytokine and chemokine expression in the GM of AIG mice

Lymphocyte migration or homing is regulated by various chemokines (23, 24), and therefore we next examined the expression profile of chemokines by RT-PCR analysis (Fig. 9). GLNs from both normal and AIG mice showed substantial expression of all chemokines assessed. As expected, CXCL13 expression was markedly augmented in the lesional GM compared with normal controls. In addition, we observed a slight augmentation of the CCL21 signal in AIG GM by RT-PCR compared with normal GM, in which weak basal expression was constantly detected (Fig. 9), although we could not detect CCL21 in the GM lymphoid clusters by immunohistochemistry (Fig. 5B). This may indicate that the CCL21 induction due to chronic inflammation outside the lymphoid cluster, probably on the tissue lymphatic endothelial cells that constitutively produce CCL21 (25), occurs in the GM of AIG mice. Another homeostatic chemokine, CCL19, was weak or undetectable, while CXCL12 was constantly detected in normal and AIG GM. The signals of inflammatory chemokines such as CCL4, CCL5, CXCL9, and CXCL10, all of which have been shown to be involved in Th1 cell migration (23), were readily detected from inflamed GM, while normal GM exhibited only faint signals. In contrast, much weaker signals compared with those from normal GLN standards were detected for Th2 attractants, CCL17 and CCL22, from the GM of both normal and AIG mice.



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  		FIGURE 9. Cytokine and chemokine mRNA profiles expressed in GLN and GM. To detect transcripts, semiquantitative RT-PCR analysis was performed. Five-fold serial dilutions of cDNA and reaction products produced without reverse transcriptase (RT-) were amplified with specific primer pairs and separated by electrophoresis. The gel images obtained are shown as the black-white reversed form. Two examples of representative AIG mice were compared with a normal mouse.

 
Gastric lesions in this disease model have been shown to constitute a Th1-type-biased, memory T cell-dependent microenvironment, as expected for the expression patterns of IFN-{gamma} and IL-4 (11) (Fig. 9). This situation is markedly different from that in regional lymph nodes, in which Th1- and Th2-type reactions are coactivated. In addition, LT-{beta}, a Th1 cytokine and key factor for the development of lymphoid organs (26, 27), is easily detected in the AIG GM, suggesting that the activated lymphocytes constitutively supply LT-{beta} throughout the chronic phase of gastric inflammation. Therefore, the pattern of cytokine and chemokine expression at each site in AIG mice well reflects effector cell localization. These findings suggest that, in contrast to the regional lymph node, the gastric lymphoid tissue of AIG mouse has the unique feature of supporting only a Th1-type immunomicroenvironment.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Tertiary lymphoid tissue is defined as a lymphoid structure induced in ectopic tissue, while the formation of secondary lymphoid tissue is developmentally programmed (2, 8, 28). In the intestine, several kinds of predetermined lymphoid tissues, such as Peyer’s patches, isolated lymphoid follicles, and crypt patches, are observed (28, 29, 30). However, under normal conditions, there is no remarkable lymphoid organization in the stomach. In this study, we found an organized lymphoid tissue associated with chronic inflammation in the GM of AIG mice. Although the sizes are relatively small (100–500 µm in diameter), each lymphoid cluster had a clear B cell follicle and T cell area with apical-basal polarity. Typical lymphoid stromal structures, such as a reticular meshwork and FDC network expressing CXCL13, support the cluster. Although the overall architecture of these lymphoid clusters was quite similar to that of secondary lymphoid tissues, several crucial differences were evident. First, the development of typical GC in the GM lymphoid follicles was quite rare. Second, naive T cells were virtually absent in the GM lesions, a finding supported by the lack of detectable expression of HEV markers (which should be required for naive T cell homing) on endothelium associated with the lymphoid clusters. Third, Th2 cell itself as well as the expression of Th2-type chemokines are negligible in the GM.

Ectopic lymphoid tissues have been reported in various diseases (2, 3, 4, 5, 6, 7). Most cases exhibit obvious B cell follicular structures with or without GC; however, T-B cell compartmentalization is less defined. Using multicolor confocal microscopy, we clearly showed T-B segregation within the GM lymphoid cluster. Therefore, similarly to within secondary lymphoid tissue, there were distinct subcompartments inside tertiary lymphoid tissue. Interestingly, GM follicles were always positioned on the luminal side of the mucosa as compared with the T cell area, suggesting that B cells and/or FDCs have some preference for the epithelial layer side.

Stromal cells expressing CXCL13 at the ectopic follicle have also been reported in human rheumatoid arthritis and Helicobacter pylori-induced follicular gastritis (3, 6), suggesting an important role of CXCL13 in the development and maintenance of the follicular structure in peripheral tissues. CXCL13 as well as lymphotoxins regulate secondary lymphoid tissue development and the proper organization of the lymphoid structures (14, 15, 26, 27). Forced expression of these factors in nonlymphoid tissue can induce neolymphoid genesis, and the constitutive production of one of these factors is sufficient for triggering the program of the lymphoid tissue development (31, 32, 33). It has also been shown that LT-{beta} plays a crucial role in the expression of CXCL13 from stromal cells (34). In the same way, LT-{beta} from long-lasting activated lymphocytes in chronic lesions should induce CXCL13 production by stromal cells and the formation of follicles or other associated lymphoid architecture. Indeed, in the AIG model, LT-{beta} expression is easily detected in the GM. In addition, as the number and size of GM lymphoid clusters become larger as the age of the mice increases, maturation of this ectopic lymphoid organization seems to depend on how long the activated lymphocytes reside in the lesion. Therefore, the findings in AIG support the above idea about the development of tertiary lymphoid tissue.

Of prime importance, the GM lymphoid tissue supports only a Th1-type response, while both Th1- and Th2-type responses are induced in the GLN (11, 35). Chemokine expression in the GM is also biased to target Th1-type effector cells as well as CXCL13 production, but not to target Th2-type effector cells. This is well in accordance with the subset localization in AIG mice. Recently, we have shown that large vessels adjacent to the lymphoid foci expressed P-selectin and mucosal addressin cell adhesion molecule-1, and possibly recruited Th1-type effector cells that showed P-selectin-ligand+ and {alpha}4{beta}7-integrinhigh phenotypes (35). Inaddition, the absence of HEV factors such as PNAd and CCL21 in vessels in the GM lymphoid tissue is likely the causative that GM-infiltrating T cells showed exclusively an activated/memory phenotype (11). Based on these findings, we speculate that the selective expression of chemoattractants and adhesion machinery in the GM lesion enables the gathering of only Th1-type-biased, activated/memory effectors, resulting in a Th1-type vicious cycle.

It is possible that the lymphoid tissue that develops in the GM of AIG mice has an important role in maintaining the autoimmune reaction with regard to autoantibody production. Pervious studies have shown that CD4+ T cells, but not CD8+ T cells or autoantibodies from AIG animals can transfer the disease to nude mice, indicating that the initiation of self reactivity is primarily mediated by CD4+ T cells (36). However, the roles of self-reactive B cells and autoantibodies in the disease progression of AIG are mostly unknown. B cells in the GM are conventional type and thus derived from the circulating pool, rather than B-1 type, which have been suggested to be self reactive (18), from the peritoneal cavity. At least two B cell subpopulations infiltrated in GM: 1) IgM+IgD+IgG- cells in the GM follicles. They are similar to those in secondary tissue follicles; therefore, they might be of naive phenotype. 2) Parafollicular plasma cells of IgG+syndecan-1+B220low phenotype. It is likely that the source of the autoantibody produced in the GM of AIG or by GM-infiltrating cells observed in vitro (11) is the latter plasma cells. Titer of serum autoantibody and the number of GM lymphoid tissue lesions actually showed a significant correlation, suggesting an important role of the ectopic follicle in the target tissue for local autoantibody production. However, typical GC morphology was rarely observed in the lymphoid tissue of the AIG stomach. This could be explained in part by the absence of Th2 cells in the lesion, because of the critical importance of a Th2 cytokine, IL-4, for mucosal GC formation (37). There is as yet no evidence supporting the notion that these plasma cells are actually differentiated from GM follicular B cells de novo. Therefore, we cannot explain the role of the GM follicular B cells at present.

IgG2a and IgG1 are the major autoantibody classes in sera (11) (unpublished data). These autoantibodies are thought to be produced in the GLN, GM, and spleen of AIG mice. Both Th1 and Th2 cells are actually activated in the GLN and spleen (11), and therefore there is a consistency between the Th subsets and Ig subclasses as a whole in this disease model. If a fraction of the plasma cells in the GM are from GM follicles in which they have matured without making clear GCs, their Ig subclass should be predominantly the Th1-supported IgG2a because of an extremely Th1-biased GM environment. However, it is also likely that the IgG1 plasma cells as well as IgG2a plasma cells that differentiated at other sites can migrate into the GM and produce IgG1 autoantibody. We have actually observed the production of both IgG1 and IgG2a autoantibodies from isolated GM cells in vitro (unpublished data). We think that in fact plasma cells may be derived from GM follicles as well as GLN.

Concerning the formation of GC, a notable finding was reported by Oshima et al. (7). They reported that a follicular gastritis characterized by large GCs in the GM was induced by the infection of neonatally thymectomized BALB/c mice with H. pylori. The pathophysiological features of H. pylori-infected AIG mice seem to be different from those of noninfected, AIG-bearing animals in several respects. For instance, H. pylori-infected AIG mice exhibited a decrease of the gland atrophy, preservation of parietal cells, and the augmentation of IL-4 expression in the GM (7). Based on these findings, we can postulate that preformed ectopic lymphoid tissue in AIG mice is a crucial prerequisite for the subsequent development of severe follicular gastritis and gastric GC induced by H. pylori infection. H. pylori-derived bacterial component(s) may alter the immunological character of the GM lymphoid tissue and the activation level of B cells with respect to the induction of mature GC. This offers the promise that GC formation in H. pylori-induced follicular gastritis could be prevented by controlling the immunological state of the tertiary lymphoid tissue.

The detailed immunological function of tertiary lymphoid tissue in the disease progression and persistence is still largely unknown. From our recent findings, additional questions emerge. Do dynamic immune reactions, such as autoantigen presentation from dendritic cells to Th1 cells, resulting in their activation and proliferation, and help for B cells to induce the affinity maturation of Ig, occur within the GM lymphoid tissue? Why does GM lymphoid tissue support only Th1-type reactions mediated by activated/memory T cells, although the tissue architecture is quite similar to that of secondary lymphoid tissue, which supports both Th1- and Th2-type reactions, as well as naive T cells? What is the true significance of the differences in the immune reactions that occur in the secondary and the tertiary lymphoid tissue?

Fortunately, the d3-Tx AIG model has several advantages for tertiary lymphoid tissue studies. For instance, because the GM lymphoid structure appears to rather strictly follow some rules, we will be able to compare in more detail the tertiary structure vs the secondary structure, including the behavior of immune cell subsets, and to evaluate subtle changes in different disease conditions. Moreover, kinetic studies of the development and maturation of tertiary lymphoid tissue could also be performed. Therefore, novel findings from the AIG model will give some clues to answering the above questions.


   Acknowledgments
 
We thank Dr. T. Masuda for useful discussions; Dr. T. Honjo for use of a confocal microscope; Drs. K. Tashiro and N. Kanazawa for PCR primers; and T. Ohfuji for excellent technical assistance.


   Footnotes
 
1 This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan. Back

2 Address correspondence and reprint requests to Dr. Akira Shimizu, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail address: shimizu{at}virus.kyoto-u.ac.jp Back

3 Abbreviations used in this paper: AIG, autoimmune gastritis; CCL, CC chemokine ligand; CR1, complement receptor-1; CXCL, CXC chemokine ligand; d3-Tx, day 3 thymectomy; FDC, follicular dendritic cell; GC, germinal center; GLN, gastric lymph node; GM, gastric mucosa; HEV, high endothelial venule; LT-{beta}, lymphotoxin-{beta}; PECAM, platelet endothelial cell adhesion molecule; PNA, peanut agglutinin; PNAd, peripheral node adressin; RN, reticular network; SLC, secondary lymphoid tissue chemokine. Back

Received for publication February 19, 2003. Accepted for publication August 12, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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