Selective accumulation of type 1 effector cells expressing P-selectin ligand and/or {alpha}4ß7-integrin at the lesions of autoimmune gastritis

Tomoya Katakai, Kazuhiro J. Mori1, Tohru Masuda2 and Akira Shimizu

Center for Molecular Biology and Genetics, Kyoto University, 53 Shogoin-kawahara-cho, Sakyo-ku, Kyoto, 606-8507, Japan
1 Department of Biology, Faculty of Science, Niigata University, Niigata 950-2181, Japan
2 The Health Examination Center, UNITIKA Central Hospital, Uji 611-0021, Japan

Correspondence to: A. Shimizu; E-mail: shimizu{at}virus.kyoto-u.ac.jp


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Th1 cells but not Th2 cells accumulate at the inflamed gastric mucosa (GM), while both subsets co-exist in the regional lymph node (RLN) in a murine experimental model for autoimmune gastritis (AIG). To understand the relationship between the immuno-microenvironment and effector localization in GM versus RLN of AIG-bearing mice, cells or tissue sections were stained with several mAb against adhesion molecules. The expression of RNA of various cytokines at these contrasting sites was also assessed. IFN-{gamma}-producing memory CD4+ (Th1) and CD8+ T cells as well as IL-12-producing mature macrophages which express P-selectin ligand and/or {alpha}4ß7-integrin selectively accumulated in the inflamed GM. Vessel endothelium at the site of infiltration expressed those counter-receptors, P-selectin and mucosal adressin cell adhesion molecule-1. Therefore, the tissue destruction of target tissue in autoimmune diseases might be promoted by a vicious circle between the selective accumulation of type 1 effectors mediated by multiple adhesion molecules and following an unusual type 1-biased microenvironment away from the type 2 response.

Keywords: CD4+ T cell subset, immuno-microenvironment, neonatal thymectomy


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Recently, it has become clear that autoreactive CD4+ T cells, especially the Th1 subset, play an important role in the development of various autoimmune diseases (1–4). Th1 and Th2 subsets of CD4+ T cells are categorized by their functions and distinct cytokine production profiles. Th1 cells induce cell-mediated (type 1) immune responses through IFN-{gamma} and lymphotoxin-ß production, while Th2 cells control humoral or allergic (type 2) responses via IL-4, -5, -10 and -13 (1,5,6). It is of prime importance that each set of cytokines shows counter-inhibitory effects against the differentiation or function of the other subset. In various situations, one of them dominates in eliciting suitable immune response according to the nature or route of entry of foreign antigens (1,5,6).

Many studies have demonstrated that the whole immune system is largely biased toward Th1-dominated (type 1) responses in experimental models of organ-specific autoimmune diseases, such as autoimmune gastritis (AIG), experimental allergic encephalomyelitis (EAE) and insulin-dependent diabetes mellitus (7–9). Among these AIG, a murine model of human type A gastritis, is induced by neonatal thymectomy 3 days after birth (d3-Tx) at high frequency especially in BALB/c mice (10). We have found that H+/K+-ATPase-specific CD4+ Th1 cells preferentially infiltrate the gastric mucosa (GM) of the corpus and possibly destroy parietal cells via Fas–Fas ligand interaction (11–13). Fas is hyper-expressed on the parietal cells in the inflamed areas of mice with AIG (AIG mice) (13). Few Th2 cells were found within the GM lesions. However, Th1 and substantial numbers of Th2 cells were found in the small curvatural lymph node [regional lymph node (RLN)] (14). Interestingly, an experimental system for transendothelial migration in vitro showed that Th1 from AIG mice rather than Th2 preferentially passed through a monolayer of endothelial cells cultured on pored filters. This result suggests that the differential migration capacity between Th1 and Th2 cells resulted in this localization of Th1 and Th2 cells in AIG (14).

These findings led us to study the mechanism of Th1 migration into the GM of AIG mice in comparison with Th2 which apparently remain in the RLN. Since CD8+ T cells and macrophages are also known to participate in immune reactions as crucial effectors and modulate the immuno-environment by secreting specific cytokines (15–17), it was also interesting to analyze their localization in AIG. In this report, we focused on the expression of two adhesion molecules, P-selectin ligand (P-sel-L) and {alpha}4ß7-integrin, on immune effector cells, and their counter-receptors, P-selectin and mucosal adressin cell adhesion molecule (MAdCAM)-1, on lesional endothelium. These receptors and their cognate ligands are involved in the initial rolling step of leukocyte extravasation (18–22). Flow cytometric and immunohistological analyses were performed to determine the expression of these adhesion molecules on infiltrating T cells and macrophages and on endothelial cells. We also detected the cytokine-producing cells by intracellular staining and flow cytometry. Cytokine gene expression was assessed by RT-PCR. Producer cells and mRNAs of IFN-{gamma}, a cytokine assigned to Th1, IL-4 and IL-10 to Th2, and IL-12 to macrophages, were detected. A close correlation between the localization of type 1 effector cells expressing the above adhesion molecules and the immuno-microenvironment in AIG is reported.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice and neonatal thymectomy
BALB/c mice were purchased from Japan SLC (Shizuoka, Japan), and maintained at the animal facility in the Center for Molecular Biology and Genetics, Kyoto University. Three days after birth, normal BALB/c mice underwent thymectomy as described previously (23). Autoimmune gastritis was diagnosed by the detection of autoantibody in sera of thymectomized mice and confirmed by histological examination. Autoantibodies were detected by ELISA using total microsomal fractions extracted from glandular stomachs of BALB/c mice as antigen (23). Goat anti-mouse IgG conjugated with horseradish peroxidase (Caltag, Burlingame, CA) and o-phenylenediamine were used as developing antibody and substrate respectively. Between 9 and 18 weeks after thymectomy, mice bearing AIG were used for experiments.

Preparation of GM-infiltrating cells
Mononuclear cells infiltrating into GM were collected as described previously (14). In brief, inflamed glandular stomachs were isolated from AIG mice and their contents washed out with HBS. Ten percent FCS-RPMI medium (RPMI 1640 supplemented with 10% FCS) was injected into the submucosa of stomachs followed by cutting up into small pieces to release infiltrating cells. After removing large tissue debris by passing through cotton in a funnel, cells were used for further experiments.

Flow cytometric analysis
Antibodies used for flow cytometric analysis were as follows. For direct staining: phycoerythrin (PE)–anti-CD4, FITC– anti-CD8 (Becton Dickinson, Mountain View, CA), FITC–anti-CD45RB, PE–anti-{alpha}4ß7, FITC–anti-Mac-1 (PharMingen, San Diego, CA), FITC–F4/80 (Caltag). For indirect staining: anti-{alpha}4ß7, biotin–anti-CD45RB (PharMingen), anti-Mac-1 (Immunotech, Marseilles, France), or culture supernatants of hybridomas: anti-CD4 (GK1.5), anti-CD8 (HO-2.2), using FITC–anti-rat IgG (Amersham, Tokyo, Japan) or biotin–anti-rat IgG (Caltag) followed by allophycocyanin (APC)–streptavidin (Caltag) as the secondary and tertiary reagents. Chimeric protein of P-selectin and human IgG1 (P-sel-Ig; PharMingen) was used to detect the functional P-sel-L by staining with biotin–anti-human IgG and APC–streptavidin or PE– streptavidin. Approximately 105–106 cells stained by the appropriate combinations of antibodies were analyzed by FACSCalibur flow cytometry and CellQuest software (Becton Dickinson). Dead cells were gated out by propidium iodide staining.

Intracellular cytokine staining
Production of IFN-{gamma}, IL-4 or IL-12 in cells was examined by an intracellular staining (24) using a Cytostain kit (PharMingen). Cells were first incubated with phorbol myristate acetate (PMA; 50 ng/ml), ionomycin (500 ng/ml) and 0.6 µl of GolgiStop (containing monensin; PharMingen) in 1 ml of 10% FCS-RPMI medium for 4 h. After blocking by normal mouse serum with 0.1 U/µl DNase I (Takara Shuzo, Otsu, Japan) to prevent non-specific binding of antibodies or cell aggregation, cells were stained with anti-CD4 or anti-CD8 antibody followed by biotin–anti-rat IgG/APC–streptavidin or anti-Mac-1 followed by Cy5–anti-rat IgG (Amersham). Cells were then fixed and permeabilized in Cytofix/Cytoperm solution (PharMingen) and stained by FITC–anti-IFN-{gamma} (Caltag) or biotin–anti-IL-12 p40 (PharMingen)/APC–streptavidin and analyzed on a FACSCalibur flow cytometer (Becton Dickinson).

Preparation and in vitro activation of P-sel-L+/– memory CD4+ T cells
Cells from spleen and lymph nodes of AIG mice were stained with anti-MHC class II (TIB120), anti-Fc{gamma}RII, III (2.4G2), anti-HSA (J11D), anti-CD8 (HO-2.2) and anti-CD45RB (PharMingen), and then plated onto a goat anti-rat IgG antibody-coated culture dish. Unbound memory CD4+ T cells were next stained with P-sel-Ig (PharMingen)/biotin–anti-rat IgG/streptavidin–MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and applied onto a MACS (Miltenyi Biotec). Column-bound or -unbound cells were used as P-sel-L+ or P-sel-L- memory CD4+ T cells respectively for in vitro stimulation with irradiated BALB/c spleen cells, anti-CD3 (145-2C11 culture supernatant, 1:100) and 1 ng/ml murine recombinant IL-12 (Genzyme, Cambridge, MA). Th1 and Th2 cells were assessed by intracellular staining for IFN-{gamma} and IL-4 expression as described previously (14).

RT-PCR
Total RNA was extracted from tissues using TRIzol Reagent (Gibco/BRL, Gaithersburg, MD) according to the manufacturer's instructions. Then 100 ng to 2 µg of RNAs was reverse transcribed by SuperScript II (Gibco/BRL) using oligo(dT) (Pharmacia Biotech, Tokyo, Japan) as primer. Specific cDNAs corresponding to reverse transcript of 0.25–1 ng of total RNAs were amplified by Taq polymerase (Takara Shuzo, Otsu, Japan) with specific primers in 25 µl reaction solutions. All PCRs were carried out with 35 cycles of denaturation (95°C, 20 s), annealing (55°C, 2 min) and extension (72°C, 1 min, with 2 s increment of each cycle), followed by polymerization (10 min, 72°C) using a DNA thermal cycler (Perkin-Elmer/Cetus, Norwalk, CT). We chose these RT-PCR conditions so that the amount of product was exponentially increasing and reflecting the content of the specific mRNA. Primers used in this study were as follows: IFN-{gamma} sense, 5'-ATCCTGCAGAGCCAGATTATC-3'; IFN-{gamma} antisense, 5'-TACTCGAGTCAGCAGCGACTCCTTT-3'; IL-4 sense, 5'-ATGGGTCCACCCCGCAGT-3'; IL-4 antisense, 5'-GCTCTTTAGGCTTTCCAGGAAGT C-3'; IL-10 sense, 5'-TCAAACAAAGGACCAGCTGGACAACATACTG-3'; IL-10 antisense, 5'-CTGTCTAGGTCCTGGAGTCCAGCAGACTCA-3'; IL-12 p40 sense, 5'-ATGTGTCCTCAGAAGCTAAC-3'; IL-12 p40 antisense, 5'-ACCAGCCATGAGCACGTGAA-3'; ß-actin sense, 5'-TCAGAAGGACTCCTATGTGG-3', ß-actin antisense, 5'-TCTCTTTGATGTCACGCACG-3'.

Immunohistochemistry
Frozen sections (10 µm) were fixed with cold acetone for 5 min, treated with PBS including 1% BSA and 10% mouse serum, and stained using the following primary antibodies: anti-P-selectin (PharMingen), anti-MAdCAM-1 (Serotec, Oxford, UK) or anti-PECAM-1 (Caltag). First antibodies were detected with PE–anti-Rat IgG (Caltag) and examined by confocal microscopy (MRC-1024; BioRad, Osaka, Japan).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Extremely type 1-biased immuno-microenvironment in the inflamed GM of AIG mice
Strong expression of mRNAs of type 1 cytokines such as IFN-{gamma} and IL-12 but weak expression of type 2 cytokines such as IL-4 and IL-10 were detected in the GM of d3-Tx AIG mice. Conversely not only type 1 but also type 2 cytokines were detected in the RLN of AIG mice (Fig. 1AGo). In addition, intracellular cytokine staining showed that not only CD4+ but also CD8+ T cells producing IFN-{gamma} markedly increased ~9- and 2-fold in the GM compared to RLN respectively (Fig. 1BGo). IL-12-producing Mac-1+ myeloid cells were also detected in the GM lesion of AIG mice, while the population with such a phenotype was rare in RLN of AIG mice as well as normal mice (Fig. 1BGo). These data suggest that the type 1 responses are dominated by effector cell accumulation in the inflamed GM, while both type 1 and 2 responses develop in RLN of AIG mice.



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 1. An extreme bias towards type 1 responses in the GM of AIG mice. (A) Strong IFN-{gamma} and IL-12 gene expression in the GM is concomitant with low or absent expression of IL-4 and IL-10. Both type 1 and 2 cytokines are expressed in the RLN of AIG mice. Total RNAs of the GM and RLN were extracted and subjected to RT-PCR analysis using specific primers. Amplified products of the expected sizes were detected after agarose gel electrophoresis. Results from one normal and two AIG mice are shown. Samples were standardized by ß-actin mRNA as an internal control. (B) Accumulation of IFN-{gamma}-producing CD4+ (upper panels) and CD8+ T cells (middle) and IL-12-producing Mac-1+ myeloid cells (lower) in the GM of AIG mice. GM-infiltrating cells and RLN cells from AIG mice or normal RLN cells were incubated with PMA/ionomycin/Golgi stop reagent for 4 h. After surface staining with anti-CD4, anti-CD8 or anti-Mac-1 (indirect staining for detection by APC or Cy5 label), cells were fixed and permeabilized. Intracellular cytokines were detected by FITC–anti-IFN-{gamma} or biotin–anti-IL-12/PE–streptavidin. Results assessed by flow cytometry are shown in dot-plots and the number in the boxed region represents the percentage within total leukocyte gated cells.

 
Accumulation of P-sel-L+ and {alpha}4ß7+ memory/effector CD4+ Th1 cells in the GM of AIG mice
Naive and memory T cells can be distinguished arbitrarily by their relative amount of CD45RB molecules expressed on cell surface (25); CD45RBhi T cells are generally naive whilst CD45RBlo represent the memory T cell population. We next examined the expression of P-sel-L and {alpha}4ß7 in either naive or memory CD4+ T cells in the GM and RLN of both normal and AIG mice. As shown in Fig. 2Go(A), P-sel-L- or {alpha}4ß7-expressing CD45RBlo cells comprise ~2–3% of the normal RLN, while these populations are elevated in the RLN and GM of AIG mice. Means of five experiments are summarized in Fig. 2Go(B); a remarkable elevation in the number of P-sel-L+ or {alpha}4ß7+ cells in the CD4+CD45RBlo population was observed especially in the GM of AIG mice.



View larger version (43K):
[in this window]
[in a new window]
 
Fig. 2. Accumulation of P-sel-L+ or {alpha}4ß7+ memory/effector CD4+ T cells in the GM of AIG mice. (A) GM-infiltrating cells and RLN cells from AIG mice or normal RLN cells were stained with anti-CD4, anti-CD45RB and P-sel-Ig (upper) or anti-{alpha}4ß7 (lower). CD4+ gated cells are shown in contour plots and the number in the boxed region represents the percentage within the CD4+ gated population. (B) The mean percentage + SD of P-sel-L+ or {alpha}4ß7+ cells within the CD4+CD45RBlo subpopulation of more than five AIG or normal mice. (C) P-sel-L+ and {alpha}4ß7+ cells among memory CD4+ T cells seem to be mutually exclusive two subsets. CD45RBlo cells from the RLN cells of AIG mice or peripheral lymph nodes (PLN) from normal mice were separated by MACS using biotin–anti-CD45RB/streptavidin–MicroBeads as unbound fraction. Separated cells were then stained by P-sel-Ig, anti-{alpha}4ß7 and anti-CD4. CD4+ gated cells are shown in contour plots. Because almost all GM-infiltrating CD4+ T cells have the CD45RBlo memory phenotype, they were not separated by MACS. (D) Accumulation of P-sel-L+ or {alpha}4ß7+ Th1 cells in the GM of AIG mice. GM-infiltrating cells and RLN cells from AIG mice were stimulated with PMA/ionomycin/Golgi stop reagent for 4 h. After staining with anti-CD4 and P-sel-Ig (upper) or anti-{alpha}4ß7 (lower), cells were fixed and permeabilized. Intra-cytoplasmic IFN-{gamma} was detected by FITC–anti-IFN-{gamma}. CD4+ gated cells are shown in dot-plots.

 
P-sel-L+ and {alpha}4ß7+ CD4+ T cells appear to be two distinct subpopulations in the GM of AIG mice, as can be seen in Fig. 2Go(C) with mutually exclusive expression of P-sel-L and {alpha}4ß7. The intracellular staining of IFN-{gamma} in combination with the surface staining of P-sel-L or {alpha}4ß7 in CD4+ cells showed that 3- to 4-fold accumulation of IFN-{gamma}-producing cells in the GM compared to the RLN were observed (Fig. 2DGo).

Dominance of Th1 cells in the splenic P-sel-L+ population of memory/effector CD4+ cells of AIG mice
We have previously shown that the majority of CD4+ cells not only in the RLN but also in spleen of AIG mice has the CD45RBlo memory/effector phenotype, and there are comparable numbers of Th1 and Th2 cells in RLN and spleen (14). Such effector CD4+ cells might be the source of GM-infiltrating cells, but among this population only Th1 cells selectively accumulated into GM. The ability to adhere to endothelial P-selectin may be a crucial difference between Th1 and Th2 cells in their migration into the inflamed tissue.

We compared Th1 and Th2 populations according to P-sel-L expression of in vitro-stimulated CD4+CD45RBlo cells from AIG spleen. In the P-sel-L+ population, which consists of about one-fifth of CD4+CD45RBlo cells in the spleen of AIG mice (data not shown), there were very few Th2 cells (IL-4 producers), but Th1 cells (IFN-{gamma} producers) dominated in this population (Table 1Go). In contrast, there is a comparable amount of Th1 and Th2 cells in the P-sel-L- population (Table 1Go) as shown previously in the total splenic CD4+ cells of AIG mice (14).


View this table:
[in this window]
[in a new window]
 
Table 1. The contents of Th1 and Th2 cells in P-sel-L+ and P-sel-L- populations of in vitro expanded memory/effector CD4+ T cells from AIG spleen.
 
Accumulation of P-sel-L+{alpha}4ß7- memory/effector CD8+ T cells into the GM of AIG mice
In contrast to CD4+ T cells, most CD8+ T cells existing not only in the normal but also in the AIG RLN, expressed naive T cell phenotype (Fig. 3Go). In the AIG RLN, only 20% of CD8+ T cells showed a CD45RBlo memory phenotype. These cells did not express P-sel-L nor {alpha}4ß7 in the normal RLN and only a small part (3%) expressed P-sel-L even in RLN of AIG mice. In marked contrast, >90% of CD8+ T cells in the GM of AIG mice were CD45RBlo memory cells. Although P-sel-L+ cells were enhanced in number 9-fold (from 3 to 27%) in the GM than those in the RLN of AIG, the concomitant expression of {alpha}4ß7 was low. These findings suggest that at least some part of memory CD8+ T cells migrate to the AIG GM using P-sel-L.



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 3. Accumulation of memory/effector CD8+ T cells of P-sel-L+ in GM of AIG mice. GM-infiltrating cells and RLN cells from AIG mice or normal RLN cells were stained with anti-CD8, anti-CD45RB and P-sel-Ig (upper) or anti-{alpha}4ß7 (lower). CD8+ gated cells are shown in contour plots.

 
Accumulation of P-sel-L+{alpha}4ß7+ mature macrophages into the GM of AIG mice
We next assessed which cell adhesion molecules are expressed by the infiltrating macrophages in the inflamed GM. Triple staining of peripheral blood leukocytes (PBL) and cells isolated from the RLN and GM of normal and AIG mice was carried out by using antibodies to Mac-1, F4/80 and either P-sel-L or {alpha}4ß7. Mac-1 detects myeloid lineage cells including monocytes/macrophages and F4/80 specifically reacts with mature macrophages.

As demonstrated in Fig. 4Go(A), an increase in the proportion of Mac-1+ cells in the PBL of AIG mice (28.9%) compared to that of normal mice (17.4%) was observed resulting from the systemic reduction of T cells caused by d3-Tx. There was also a significant number of Mac-1+ cells in the total GM infiltrates of AIG mice (11.4%). However, such Mac-1+ cells were a rare population (1.2%) in the RLN of AIG mice. Interestingly, within the Mac-1+ population detected in the PBL, ~11% were double-positive for P-sel-L and {alpha}4ß7; little increase of this population from normal was observed in the PBL of AIG mice (Fig. 4BGo). In contrast, selective accumulation of P-sel-L+{alpha}4ß7+ cells in Mac-1+ cells was attained in the GM of AIG mice. Such cells in the GM were increased 3-fold compared to those in the PBL (Fig. 4BGo). Mac-1+{alpha}4ß7+ cells observed in the GM were mostly P-sel-L+ as well as F4/80+, in addition, producing IL-12. PBL Mac-1+{alpha}4ß7+ cells were F4/80lo and did not produce IL-12 (Fig. 4B and CGo). These findings indicate that PBL include Mac-1+P-sel-L+{alpha}4ß7+ monocytes and this population can extravasate to the inflamed GM but not to the regional lymph node from the circulation to mature into IL-12-producing macrophages.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 4. Accumulation of mature macrophages of P-sel-L+{alpha}4ß7+ in the GM of AIG mice. (A and B) GM-infiltrating cells, RLN and PBL from AIG mice or normal mice were stained with anti-Mac-1, P-sel-Ig and anti-{alpha}4ß7 (A and B, upper) or anti-Mac-1, anti-{alpha}4ß7 and anti-F4/80 (B, lower). Total leukocyte gated (A) or Mac-1+ gated (B) cells are shown in histograms or contour plots respectively. (C) PBL, RLN and GM-infiltrating cells of AIG mice were stimulated with PMA/ionomycin/Golgi stop reagent for 4 h. After staining cells with anti-Mac-1 and anti-{alpha}4ß7, the cells were fixed and permeabilized. Intracellular IL-12 was detected by biotin–anti-IL-12 p40 followed by APC–streptavidin. Mac-1+ gated cells are shown in dot-plots.

 
Detection of P-selectin and MAdCAM-1 on vessel endothelium at the site of inflammation
Serial tissue sections prepared from normal and AIG stomachs were stained with hematoxylin or antibodies against endothelial adhesion receptors, P-selectin, MAdCAM-1 or PECAM-1, a general marker of blood vessels (26). In contrast to the normal stomach, gland atrophy was seen in the GM of AIG mice, in association with severe mononuclear cell infiltration in the lamina propria (Fig. 5A and BGo, arrows). PECAM-1 was detected on all small and large vessels in both normal and AIG mice (Fig. 5G and HGo). Large vessels in the lamina propria of AIG mice adjacent to the sites of lymphocyte infiltration were more strongly stained for P-selectin and MAdCAM-1 compared to normal mice (Fig. 5D and FGo, arrows). In particular, expression of P-selectin and MAdCAM-1 in the normal glandular mucosa was weak and mostly limited to a small number of vessels distributed throughout the lamina propria of the crypts (Fig. 5C and EGo).



View larger version (39K):
[in this window]
[in a new window]
 
Fig. 5. Expression of P-selectin and MAdCAM-1 on the lesional endothelium of AIG GM. Serial frozen sections of stomachs from normal (left panels) or AIG (right panels) were stained with anti-P-selectin (C and D), anti-MAdCAM-1 (E and F), anti-PECAM-1 (G and H) or control rat IgG (I and J) followed by PE–anti-rat IgG. Hematoxylin staining is shown in (A) and (B). Endothelium adjacent to infiltrating mononuclear cells was strongly positive for P-selectin and MAdCAM-1 (arrows) (original magnification x200).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study, we demonstrated an extreme type 1-biased autoimmune lesion accompanied by the selective accumulation of characteristic effector cells including CD4+ Th1 cells, CD8+ cells and macrophages in the GM of AIG mice. This sharply contrasts to the RLN in which both type 1 and 2 immune reactions occurred. Previous studies have suggested an indispensable role for autoreactive Th1 cells in this model system (11,12,27). As also shown in many other models, it is clear that Th1 cells are central effectors for the development of organ-specific autoimmunity (8,9), though what actually initiates or triggers such reactions is not known yet. The tendency toward the type 1 response induced by d3-Tx (28) might promote the triggering of self-reactivity accompanied by an impaired development of regulatory CD4+ T cells that can block the disease (29). However, we have found a substantial number of Th2 cells in the RLN of AIG mice (14). Th2 cells are thought to regulate the cytotoxic or tissue destructive type 1 response via inhibition of Th1 development under physiological conditions. Th2 activation together with the expression of the immuno-suppressive cytokine, IL-10 (30,31), would support the idea that type 2 responses compete with the type 1 responses, thereby balancing the responses within the RLN of AIG mice.

In inflamed GM, in marked contrast to RLN, there are very few Th2 cells. However, IFN-{gamma}-producing CD8+ and IL-12-producing macrophages and Th1 cells had accumulated. MHC class II (13,33) and Fas (13) molecules are ectopically expressed on the parietal cells of AIG mice. Therefore, in addition to forming the biased immune environment towards type 1, these accumulated effectors may induce apoptosis of parietal cells by several different ways including direct interactions between Fas and Fas ligand or with perforin and through soluble mediators such as tumor necrosis factor-{alpha} or lymphotoxins. Local production of IL-12 by myeloid lineages may strongly support Th1 and cytotoxic CD8+ T cell functions in this area. CD8+ T cells were a major population of IFN-{gamma}-producers in the GM lesions similar to Th1 cells. Therefore, they could contribute to promoting the disease progression by forming a lesional type 1 environment as well as by tissue injury by the helps of Th1 cells or IL-12 derived from macrophage, even though they alone could not initiate the AIG (32). Thus, memory CD8+ T cells may have important roles in the exacerbation of this disease.

We also showed that effector cells expressing P-sel-L or {alpha}4ß7-integrin selectively accumulated in the inflamed GM. P-selectin expressed on activated endothelium is a crucial molecule in the recruitment of immune cells to inflamed tissue (20,34). Recently, selective expression of P-sel-L on Th1 but not on Th2 cells, their mediation of the selective infiltration of Th1 into the inflamed tissue and an enhancement by IL-12 of P-selectin binding by Th1 and CD8+ cells were shown (35–37). These reports are consistent with our findings that the accumulation of P-sel-L+ effector T cells into the inflamed GM and the expression of P-selectin on the lesional endothelium. Since P-sel-L on T cells is restricted to CD45RBlo memory cells, the P-selectin/P-sel-L system is also thought to recruit memory/effector T cells effectively to sites of inflammation. Most in vitro expanded P-sel-L+CD45RBloCD4+ cells from the AIG spleen were Th1 but not Th2 cells. Therefore, attachment of effector/memory T cells to the blood vessel endothelium using these adhesion molecules, as an initial step of transmigration, might promote the preference of Th1 cells in the inflamed lesion. As a ligand for MAdCAM-1, {alpha}4ß7-integrin mediates lymphocyte homing to mucosal tissues (21,22). Ectopic expression of MAdCAM-1 on the endothelium at chronic inflammation (38,39) suggests that activated immune effector cells may infiltrate these sites using {alpha}4ß7-integrin/MAdCAM-1. The high level of expression of {alpha}4ß7-integrin in a subset of memory CD4+ T cells suggests its involvement in memory T cell recruitment. In accordance with this idea, we have shown the accumulation of memory/effector CD4+ T cells expressing {alpha}4ß7-integrin and that MAdCAM-1 is expressed on endothelium in the inflamed GM. A considerable fraction of IFN-{gamma}-producing CD4+ T cells in the GM showed P-sel-L+ or {alpha}4ß7+ phenotypes. These observations suggest both of the P-selectin/P-sel-L and {alpha}4ß7-integrin/MAdCAM-1 adhesion systems may play significant roles in the accumulation of type 1 effector cells at inflamed GM of AIG.

It is thought that immune effector cells have specific sites of activity and migrate to such sites via unique mechanisms (18,19). In accidental infection, local type 1-biased immune response by the rapid accumulation of cytotoxic effector cells to the infected site effectively eliminates pathogens. However, this becomes a dangerous system if the targets are the self-antigens presented over a long period. Thus, we suggest that, in chronic organ-specific autoimmunity, the selective migration of type 1 effectors into the target tissue isolated from type 2 or regulatory populations causes an unusual bias toward type 1 immunity and accelerates further self-tissue destruction, even though Th2 cells are in draining lymph node. There are preferences in the numerous steps of extravasation of these type 1 effector cells compared to type 2 cells. Such preferences are seen in the initial attachment using specific adhesion systems (shown here), in the passing step through endothelial cells (14) and also in the attraction by specific chemokines (our preliminary results using RT-PCR to detect mRNAs, data not shown). The preference in each step is not absolute but probably acts in a cumulative manner and finally forms a strong bias to type 1 immunity. Similar vicious circles between the microenvironment and the recruitment of immune cells might be formed in various kinds of autoimmune inflammation. Breaking such feedback loops by promoting or preventing the migration of particular cell types is a promising target for developing novel therapies.


    Acknowledgments
 
We are grateful to Drs Y. Agata, M. Sugai, E. Matsuda and H. Gonda for useful suggestions and discussion, and to Dr S. Fraser for critical reading of this manuscript. We also thank Dr S. Hirano for excellent technical assistance. This work was supported in part by a grant-in aid from the Ministry of Education, Science and Culture of Japan.


    Abbreviations
 
AIG autoimmune gastritis
APC allophycocyanin
d3-Tx day 3 thymectomy
GM gastric mucosa
MAdCAM mucosal adressin cell adhesion molecule
PBL peripheral blood leukocytes
PE phycoerythrin
PMA phorbol myristate acetate
P-sel-L P-selectin ligand
RLN regional lymph node

    Notes
 
Transmitting editor: T. Kurosaki

Received 11 August 2001, accepted 23 October 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Abbas, A. K., Murphy, K. M. and Sher, A. 1996. Functional diversity of helper T lymphocytes. Nature 383:787.[ISI][Medline]
  2. Liblau, R. S., Singer, S. M. and McDevitt, H. O. 1995. Th1 and Th2 CD4+ T cells in the pathogenesis of organ-specific autoimmune diseases. Immunol. Today 16:34.[ISI][Medline]
  3. Powrie, F. and Coffman, R. L. 1993. Cytokine regulation of T-cell function: potential for therapeutic intervention. Immunol. Today 14:270.[ISI][Medline]
  4. Röcken, M. and Shevach, E. M. 1996. Immune deviation—the third dimension of nondeletional T cell tolerance. Immunol. Rev. 149:175.[ISI][Medline]
  5. Paul, W. E. and Seder, R A. 1994. Lymphocyte responses and cytokines. Cell 76:241.[ISI][Medline]
  6. Seder, R. A. and Paul, W. E. 1994. Acquisition of lymphokine-producing phenotype by CD4+ T cells. Annu. Rev. Immunol. 12:635.[ISI][Medline]
  7. Gleeson, P. A., Toh, B.-H. and van Driel, I. R. 1996. Organ-specific autoimmunity induced by lymphopenia. Immunol. Rev. 149:97.[ISI][Medline]
  8. Miller, S. D. and Karpus, W. J. 1994. The immunopathogenesis and regulation of T-cell-mediated demyeliniating diseases. Immunol. Today 15:356.[ISI][Medline]
  9. Delovitch, T. L. and Singh, B. 1997. The nonobese diabetic mouse as a model of autoimmune diabetes: immune dysregulation gets the NOD. Immunity 7:727.[ISI][Medline]
  10. Kojima, A. and Prehn, R. T. 1981. Genetic susceptibility of post-thymectomy autoimmune diseases in mice. Immunogenetics 14:15.[ISI][Medline]
  11. Nishio, A., Hosono, M., Watanabe, Y., Sakai, M., Okuma, M. and Masuda, T. 1994. A conserved epitope on H+,K+-adenosine triphosphatase of parietal cells discerned by a murine gastritogenic T-cell clone. Gaseroenterology 107:1408.
  12. Katakai, T., Agata, Y., Shimizu, A., Ohshima, C., Nishio, A., Inaba, M., Kasakura, S., Mori, K. J. and Masuda, T. 1997. Structure of T cell receptor expressed on a gastritogenic T cell clone, II-6, and frequent appearance of similar clonotypes in mice bearing autoimmune gastritis. Int. Immunol. 9:1849.[Abstract]
  13. Nishio, A., Katakai, T., Oshima, C., Kasakaura, S., Sakai, M., Yonehara, S., Suda, T., Nagata, S. and Masuda, T. 1996. A possible involvement of Fas–Fas ligand signaling in the pathogenesis of murine autoimmune gastritis. Gastroenterology 111:959.[ISI][Medline]
  14. Katakai, T., Mori, K. J., Masuda, T. and Shimizu, A. 1998. Differential localization of Th1 and Th2 cells in autoimmune gastritis. Int. Immunol. 10:1325.[Abstract]
  15. Harty, J. T., Tvinnereim, A. R. and White, D. W. 2000. CD8+ T cell effector mechanisms in resistance to infection. Annu. Rev. Immunol. 18:275.[ISI][Medline]
  16. Cerwenka, A., Morgan, T. M, Harmsen, A. G. and Dutton, R. W. 1999. Migration kinetics and final destination of type 1 and type 2 CD8 effector cells predict protection against pulmonary virus infection. J. Exp. Med. 189:423.[Abstract/Free Full Text]
  17. Jun, H.-S., Yoon, C.-S., Zbytnuik, L., van Rooijen, N. and Yoon, J.-W. 1999. The role of macrophages in T cell-mediated autoimmune diabetes in nonobese diabetic mice. J. Exp. Med. 189:347.[Abstract/Free Full Text]
  18. Springer, T. A. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76:301.[ISI][Medline]
  19. Butcher, E. C., Williams, M., Youngman, K., Rott, L. and Briskin, M. 1999. Lymphocyte trafficking and regional immunity. Adv. Immunol. 72:209.[ISI][Medline]
  20. Mayadas, T. N., Johnson, R. C., Rayburn, H., Hynes, R. O. and Wagner, D. D. 1993. Leukocyte rolling and extravasation are severely compromised in P selectin-deficient mice. Cell 74:541.[ISI][Medline]
  21. Berlin, C., Berg, E. L., Briskin, M., Andrew, D. P., Kilshaw, P. J., Holzmann, B., Weissman, I. L., Hamann, A. and Butcher, E. C. 1993. {alpha}4ß7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1. Cell 74:185.[ISI][Medline]
  22. Berlin, C., Bargatze, R. F., Campbell, J. J., von Andrian, U. H., Szabo, M. C., Hasslen, S. R., Nelson, R. D., Berg, E. L., Erlandsen, S. L. and Butcher, E. C. 1995. {alpha}4 integrins mediate lymphocyte attachment and rolling under physiologic flow. Cell 80:413.[ISI][Medline]
  23. Fukuma, K., Sakaguchi, S., Kuribayashi, K., Chen, W. L., Morishita, R., Sekita, K., Uchino, H. and Masuda, T. 1988. Immunologic and clinical studies on murine experimental autoimmune gastritis induced by neonatal thymectomy. Gastroenterology 94:274.[ISI][Medline]
  24. Openshaw, P., Murphy, E. E., Hosken, N. A., Maino, V., Davis, K., Murphy, K. and O'Garra, A. 1995. Heterogeneity of intracellular cytokine synthesis at the single-cell level in polarized T helper 1 and T helper 2 populations. J. Exp. Med. 182:1357.[Abstract]
  25. Croft, M., Duncan, D. D. and Swain, S. L. 1992. Response of naive antigen-specific CD4+ T cells in vitro: characteristics and antigen-presenting cell requirements. J. Exp. Med. 176:1431.[Abstract]
  26. DeLisser, H. M., Newman, P. J. and Albelda, S. M. 1994. Molecular and functional aspects of PECAM-1/CD31. Immunol. Today 15:490.[ISI][Medline]
  27. Barrett, S. P., Gleeson, P. A., de Silva, H., Toh, B.-H. and van Driel, I. R. 1996. Interferon-{gamma} is required during the initiation of an organ-specific autoimmune disease. Eur. J. Immunol. 26:1652.[ISI][Medline]
  28. Adkins, B. and Du, R.-Q. 1998. Newborn mice develop balanced Th1/Th2 primary effector responses in vivo but are biased to Th2 secondary responses. J. Immunol. 160:4217.[Abstract/Free Full Text]
  29. Asano, M., Toda, M., Sakaguchi, N., Sakaguchi, S. 1996. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J. Exp. Med. 184:387.[Abstract]
  30. Strdeur, P. and Goldman, M. 1998. Interleukin-10 as a regulatory cytokine induced by cellular stress: molecular aspects. Int. Rev. Immunol. 16:501.[Medline]
  31. Liu, L., Rich, B. E., Inobe, J., Chen, W. and Weiner, H. L. 1998. Induction of Th2 cell differentiation in the primary immune response: dendritic cell isolated from adherent cell culture treated with IL-10 prime naive CD4+ T cells to secrete IL-4. Int. Immunol. 10:1017.[Abstract]
  32. Sakaguchi, S., Fukuma, K., Kuribayashi, K. and Masuda, T. 1985. Organ-specific autoimmune diseases induced in mice by elimination of T cell subset. I. Evidence for the active participation of T cells in natural self-tolerance; deficit of a T cell subset as a possible cause of autoimmune disease. J. Exp. Med. 161:72.[Abstract]
  33. Martinelli, T. M., van Driel, I. R., Alderuccio, F., Gleeson, P. A. and Toh, B.-H. 1996. Analysis of mononuclear cell infiltrate and cytokine production in murine autoimmune gastritis. Gastroenterology 110:1791.[ISI][Medline]
  34. Varki, A. 1994. Selectin ligands. Proc. Natl. Acad. Sci. 91:7390.[Abstract]
  35. Austrup, F., Vestweber, D., Borges, E., Löhning, M., Bräuer, R., Herz, U., Renz, H., Hallmann, R., Scheffold, A., Radbruch, A. and Hamann, A. 1997. P- and E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells into inflamed tissues. Nature 385:81.[ISI][Medline]
  36. Tietz, W., Allemand, Y., Borges, E., von Laer, D., Hallmann, R., Vestweber, D. and Hamann, A. 1998. CD4+ T cells migrate into inflamed skin only if they express ligands for E- and P-selectin. J. Immunol. 161:963.[Abstract/Free Full Text]
  37. Xie, H., Lim, Y.-C., Luscinskas, F. W. and Lichtman, A. H. 1999. Acquisition of selectin binding and peripheral homing properties by CD4+ and CD8+ T cells. J. Exp. Med. 189:1765.[Abstract/Free Full Text]
  38. Faveeuw, C., Gagnerault, M.-C. and Lepault, F. 1994. Expression of homing and adhesion molecules in infiltrated islets of Langerhans and salivary glands of nonobese diabetic mice. J. Immunol. 152:5969.[Abstract/Free Full Text]
  39. Picarella, D., Hurlbut, P., Rottman, J., Shi, X., Butcher, E. C. and Ringler, D. J. 1997. Monoclonal antibodies specific for ß7 integrin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) reduce inflammation in the colon of scid mice reconstituted with CD45RBhigh CD4+ T cells. J. Immunol. 158:2099.[Abstract]