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Arthritis Rheum. Author manuscript; available in PMC 2009 Nov 25.
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PMCID: PMC2782777
NIHMSID: NIHMS146217

Development of Autoimmune Hepatitis-like Disease and Autoantibody Production to Nuclear Antigens in Mice Lacking B and T Lymphocyte Attenuator (BTLA)

Abstract

Objective

B and T lymphocyte attenuator (BTLA), a coreceptor expressed on lymphocytes, has recently been described as an inhibitory coreceptor that negatively regulates lymphocyte activation. The purpose of this study was to investigate the role of BTLA in the regulation of immune homeostasis and the pathogenesis of autoimmunity.

Methods

We examined the levels of immunoglobulins, autoantibodies to nuclear antigens, and activation status of T cells in BTLA-deficient (BTLA−/−) mice. We also examined histopathologic changes of the organs in BTLA−/− mice.

Results

We found that BTLA−/− mice gradually developed hyper-γ-globulinemia, antinuclear antibody, anti-SS-A antibody and anti-double-strand DNA antibody, and an increase of activated CD4+ T cells in the periphery with age. Lack of BTLA led to spontaneous development of autoimmune hepatitis (AIH)-like disease characterized by elevation of transaminases and interface hepatitis and spotty necrosis in the liver. BTLA−/− mice also showed inflammatory cell infiltration in multiple organs including salivary glands, lungs and pancreas, similar to Sjögren’s syndrome, a frequent complication of AIH. Furthermore, BTLA−/− mice showed a significant reduction in the survival rate after the age of 7 months.

Conclusion

Our results indicate that BTLA plays an important role in the maintenance of immune tolerance and the prevention of autoimmune diseases.

Introduction

Accumulating evidence indicates that signals through the inhibitory coreceptors such as CTLA-4 and PD-1 are crucial in regulating T cell activation (13). CTLA-4-deficient mice rapidly develop a lymphoproliferative disease with multiorgan lymphocytic infiltration and tissue destruction, with severe myocarditis and pancreatitis, and die by 3–4 weeks of age (4). PD-1-deficient mice on a C57BL/6 background spontaneously develop lupus-like glomerulonephritis and proliferative arthritis (5). In addition, PD-1-deficient mice on a BALB/c background develop dilated cardiomyopathy (6), which is associated with the production of autoantibody against cardiac troponin I (7), and die of congestive heart failure. These results strongly suggest that inhibitory coreceptors play crucial roles in maintaining immune homeostasis and thus inhibiting autoimmunity.

We have recently identified the third inhibitory coreceptor, B and T lymphocyte attenuator (BTLA/CD272), which is a cell surface molecule expressed on lymphocytes and some of non-lymphoid hematopoietic cells, and exhibits similarities to CTLA-4 and PD-1 (8, 9). BTLA interacts with a TNF receptor family member herpesvirus entry mediator (HVEM) (1012) and the ligation of BTLA induces tyrosine phosphorylation of its immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and SHP-1/SHP-2 association and then attenuates IL-2 production and T cell proliferation (8, 13). Moreover, we found that sensitivity to experimental autoimmune encephalomyelitis as well as antigen-specific antibody responses were enhanced in BTLA-deficient (BTLA−/−) mice (8). These data indicate that, like CTLA-4 and PD-1, BTLA negatively regulates immune responses. However, the role of BTLA in the protection from autoimmune diseases remains to be determined.

In the present study, we investigated the role of BTLA in the regulation of immune homeostasis and the pathogenesis of autoimmunity. We found that the levels of immunoglobulins and autoantibodies to nuclear antigens were gradually elevated with age in BTLA−/− mice. In the periphery, activated CD4+ T cells were gradually accumulated in BTLA−/− mice. Moreover, autoimmune hepatitis (AIH)-like disease developed spontaneously in BTLA−/− mice, which was associated with the increase in activated CD4+ T cells and NK T cells in the liver. Furthermore, infiltration of inflammatory cells was observed in multiple organs such as salivary glands, lungs and pancreas in BTLA−/− mice. Our results indicate that BTLA plays an important role in the immune surveillance against autoimmune diseases.

Materials and Methods

Mice

BTLA-deficient (BTLA−/−) mice were established on a 129SvEv background as described previously (8). Mice were housed in microisolator cages under specific pathogen-free conditions. Gender-matched littermate heterozygous or wild-type (WT) mice were bled with BTLA−/− mice in the same cage until analysis and used as controls. All experiments were performed according to the guidelines of Chiba University.

Antibodies

The following antibodies were purchased from BD PharMingen (Franklin Lakes, NJ): anti-CD3e biotin, PE (145-2C11), anti-CD4 FITC, PE (L3T4, RM4-5), anti-CD8α FITC, PE, Cy-chrome (Ly-2, 53-6.7), anti-CD25 biotin (PC61), anti-CD45R/B220 FITC, PE (RA3-6B2), anti-CD69 FITC (H1.2F3), anti-CD62L PE (MEL-14), anti-Ly-6G/C PE (Gr-1), anti-CD11c FITC (HL3), anti-CD11b (Mac-1) PE (M1/70), anti-CD49b/Pan-NK PE (DX5), anti-CD122 biotin (TM-β1), anti-TCR Vβ3 (KJ25)/Vβ5 (MR9-4)/Vβ6 (RR4-7)/Vβ8 (F23.1)/Vβ10 (B21.5)/Vβ11 (RR3-15) FITC, anti-IL-4 PE (BVD4-1D11), anti-IFNγ FITC (XMG1.2) and APC-Streptavidin (SA). Anti-Foxp3 FITC (FJK-16s) was purchased from eBioscience (San Diego, CA).

Flow cytometric analysis

Single cell suspensions of thymocytes, splenocytes and mononuclear cells in the liver were obtained from 12-month-old BTLA−/− mice and littermate WT mice. The mononuclear cells in the liver were separated by Ficoll gradient fractionation. The cells (1 × 106) were washed twice, stained with antibodies described above, and analyzed on a FACSCalibur (Becton Dickinson, Mountain View, CA) using CELLQuestsoftware (Becton Dickinson). Prior to staining, Fc receptors were blocked with anti-CD16/32 antibody (2.4G2, BD PharMingen). Intracellular cytokine staining for IL-4, IFN-γ and Foxp3 was performed as described previously (14).

Cell culture

Splenocytes from 12-month-old WT mice and littermate BTLA−/− mice were stimulated with plate-bound anti-CD3ε mAb (5 μg/ml; 145-2C11) in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 50 μM 2-ME and 2 mM L-glutamine in a 24-well microtiter plate at 37°C for 48 h.

ELISA

Culture supernatants were collected and the levels of IFN-γ, IL-4, IL-5 (BD PharMingen) and IL-17 (R&D Systems, Minneapolis, MN) in supernatants were measured by ELISA kits according to the manufacturer’s protocol. The minimum significant values were 15 pg/ml for IL-4, IL-5 and IL-17 and 30 pg/ml for IFN-γ.

Histopathological Analysis

Lung, heart, liver, pancreas, thymus, spleen, lymph node, kidney, stomach, small and large intestine, salivary glands, skin and joints were subjected to histological examination. Tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and embedded in paraffin. Sections (3 μm thick) were stained with hematoxylin and eosin (HE) and examined by microscopy. Liver sections were also stained with reticulum fiber silver stain by standard protocols. Kidney sections were stained with periodic acid methenamine silver (PAM) stain and periodic-acid Schiff (PAS). A semi-quantitative scoring method was applied to grade the magnitude of inflammatory cell infiltration in tissue as described previously (15). In brief, the numbers of small foci (30–99 cells/focus) and large foci (100 cells ~/focus) per cm2 were counted on HE-stained sections by a pathologist in a blinded manner.

Measurement of serum immunoglobulins, autoantibodies and biochemical analysis of sera

Sera were obtained from individual BTLA−/− mice and littermate WT mice periodically at 3-month-intervals from 3 to 15 months of age and the levels of IgM, IgG1, IgG2a, IgG2b and IgG3 were determined by enzyme-linked immunosorbent assay (ELISA) using SBA Clonotyping System/HRP kits (Southern Biotechnology Associates, Inc., Birmingham, AL) following manufacturer’s instruction. The levels of anti-nuclear antibody (ANA) in serum were determined by using an ELISA kit (MESACUP ANA kit, MBL, Nagoya, Japan), which contains human RNP, SS-B, Scl-70, Jo-1, CENP-B, Ribosomal P, DNA and histones and bovine Sm and SS-A as nuclear antigens, with horseradish peroxidase-conjugated anti-mouse IgG antibody (Amersham Bioscience, UK). The levels of anti-dsDNA antibody and anti-SS-A antibody were determined by using MESACUP DNA-II Test-ds kit (MBL) and MESACUP-2 Test SS-A kit (MBL), respectively (16). Pooled serum from 4-month-old MRL-lpr/lpr mice (Charles River Laboratories, Atsugi, Japan) was used as a positive control. The levels of asparate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamic transpeptidase (γ–GTP), alkaline phosphatase (ALP), total bilirubin (T-Bil) and blood urea nitrogen (BUN) were determined by standard protocols (SRL, Inc., Tokyo, Japan).

Statistical analysis

Data are summarized as mean ± SD. The unpaired t-test was used for statistical analysis. P values less than 0.05 were considered significant.

Results

Hyper-γ-globulinemia and autoantibodies to nuclear antigens in BTLA−/− mice

To define the role of BTLA in the regulation of immune homeostasis and the pathogenesis of autoimmunity, we first assessed the levels of immunoglobulins in BTLA−/− mice (8) and littermate wild-type (WT) mice. BTLA−/− mice showed no apparent abnormalities by the age of 3 months. However, the levels of IgG1 and IgG2a were gradually increased in BTLA−/− mice and became significantly higher in BTLA−/− mice at 7 months of age than those in WT mice (IgG1: BTLA−/− mice 3,831 ± 1,065 vs. WT mice 1,318 ± 402 μg/ml, mean ± SD, n = 4–6, p<0.001) (IgG2a: BTLA−/− mice 21,240 ± 8,931 vs. WT mice 11,080 ± 4,931 μg/ml, p<0.03) (Figure 1A). In contrast, no significant difference was observed in the levels of IgM, IgG2b and IgG3 between BTLA−/− mice and WT mice (Figure 1A). Importantly, the levels of anti-nuclear antibody (ANA) and anti-double-strand DNA (anti-dsDNA) antibody were also gradually increased in BTLA−/−mice and became significantly higher in BTLA−/− mice at 9 and 12 months of age as compared with those in age-matched WT mice (n = 11–21, p<0.05 to 0.001) (Figure 1B). We also found that anti-SS-A/Ro antibody, an autoantibody often found in patients with Sjögren’s syndrome, was significantly increased in BTLA−/− mice at 12 months of age (Figure 1B). The frequencies of the elevated titers of ANA, anti-dsDNA antibody and anti-SS-A antibody were 67% (8/12), 55% (11/20) and 75% (12/16), respectively, in BTLA−/− mice at 12 months of age (Figure 1B).

Figure 1
Elevated immunoglobulins and autoantibodies to nuclear antigens in BTLA−/− mice

Activation of CD4+ T cells and breakdown of self-tolerance in BTLA−/− mice

Since the observations described above suggest that the BTLA deficiency may result in the breakdown of self-tolerance, we then examined the activation state of T cells in periphery in BTLA−/− mice. The number of splenocytes in 12-month-old BTLA−/− mice was increased as compared with that in WT mice by approximately 70% (BTLA−/− mice 52.6 ± 4.4 vs. WT mice 31.5 ± 3.4, × 106, n = 4, p<0.05) (Figure 2A). FACS analysis revealed that the numbers of CD4+ T cells and B cells were significantly increased in 12-month-old BTLA−/− mice as compared with those in WT mice (n = 4, p<0.05) (Figure 2A). In contrast, the number of whole DCs (CD11c+ cells) as well as the frequency of each DC subtype, namely CD11b+ myeloid DCs, CD8+ lymphoid DCs and plasmacytoid DCs, was indistinguishable between BTLA−/− mice and WT mice (data not shown). CD4+ T cells in the spleen of BTLA−/− mice expressed higher levels of CD25 and CD69 and a lower level of CD62L than WT CD4+ T cells (Figure 2B). However, the frequency of Foxp3-expressing CD4+ T cells in 12-month-old BTLA−/− mice was not increased (Figure 2B), suggesting that the increase of CD25+ CD4+ T cells is accounted for by the increase of preactivated conventional CD4+ T cells but not of naturally occurring CD4+ CD25+ regulatory T cells. CD8+ T cells also tended to express higher levels of CD25 and a lower level of CD62L in 12-month-old BTLA−/− mice (Figure 2B). Consistent with the preactivated state of CD4+ T cells in BTLA−/− mice, splenocytes from BTLA−/− mice produced 2 to 3 fold higher levels of IFNγ, IL-4 and IL-5 in response to anti-CD3 stimulation (Figure 2C). Intracellular cytokine staining of anti-CD3-stimulated splenocytes revealed that CD4+ T cells were a major source of IL-4 and IFNγ in BTLA−/−mice (Figure 2C). On the other hand, IL-17 production from anti-CD3-stimulated splenocytes in BTLA−/− mice was similar to that in WT mice (Figure 2C).

Figure 2
Flow cytometric analysis of splenocytes and thymocytes from BTLA−/− mice

To test whether the impairment of thymic selection is involved in the activation of peripheral T cells in BTLA−/− mice, we analyzed thymocytes in 12-month-old BTLA−/−mice. Consistent with previous reports that examined the thymi of young BTLA−/− mice (8, 9), there is no gross abnormality in thymus. The total cell number of thymus in 12-month-old BTLA−/− mice was comparable to that in WT mice (22.7 ± 3.7 vs. 19.0 ± 5.7, × 106, n = 4) (Figure 2D). The numbers of CD4+CD8+ double-positive thymocytes, CD4+CD8 thymocytes and CD4CD8+ thymocytes in 12-month-old BTLA−/− mice were also similar to those of WT mice (Figure 2D). The expression levels of CD3ε and CD69 during thymocyte differentiation were also normal in BTLA−/− mice (Figure 2E).

Next, we analyzed clonal deletion of thymocytes expressing specific TCR Vβ families (17, 18) in 12-month-old BTLA−/− mice. The frequency of CD4+CD8 thymocytes expressing TCR Vβ3 is known to be very low in the 129 strain (19). We found that the frequency of CD4+CD8 thymocytes expressing TCR Vβ3 was similarly low in BTLA−/−mice and WT mice as compared with that in C57BL/6 mice (Figure 2F). Frequencies of the other TCR Vβ families in CD4+CD8 thymocytes in BTLA−/− mice were also comparable to those in WT mice (Figure 2F). Moreover, we confirmed that the clonal deletion of Vβ5, Vβ11 and Vβ12-expressing CD4+CD8 thymocytes also occurred normally in BTLA−/− mice on a BALB/c background (data not shown). These data suggest that thymic selection is not impaired in BTLA−/− mice and thus that CD4+ T cells in BTLA−/− mice are spontaneously and polyclonally activated in the periphery.

Spontaneous development of autoimmune hepatitis-like disease in BTLA−/− mice

We next examined the organs histologically in 12-month-old BTLA−/− mice. Interestingly, among the organs examined, liver showed prominent inflammatory changes in BTLA−/− mice. Liver sections from BTLA−/− mice showed a mononuclear cell infiltration in the portal tracts (Figure 3D, E) of which the inflammatory infiltrates disrupted the limiting plate and penetrated into the periportal hepatic lobule (Figure 3D, E, G). This inflammatory cell infiltration extending into the adjacent parenchyma (interface hepatitis or piecemeal necrosis) is thought to be a hallmark of autoimmune hepatitis (AIH) in humans (20). Inflammatory infiltrates were predominantly composed of blastic lymphocytes and an affluent number of plasma cells (Figure 3H) with occasional eosinophils (Figure 3I) and neutrophils. In addition to portal areas, spotty or patchy inflammatory cell infiltration was observed within the lobules. These multilobular foci were associated with degeneration of the hepatocytes (spotty necrosis) (Figure 3F).

Figure 3
Spontaneous development of autoimmune hepatitis-like disease in BTLA−/− mice

Liver sections of 12-month-old BTLA−/− mice also showed the prominent subendothelial mononuclear cell infiltration in the portal and hepatic veins that resulted in the detachment of endothelial cells from the basement membrane (endothelialitis) (Figure 3J). Bile ducts were also damaged by severe inflammation. The epithelial cells of the bile ducts showed nuclear pleomorphism, vacuolation of cytoplasm, cell stratification or loss, and irregularity of duct outlines (Figure 3K, L). Taken these pathological findings together with elevated immunoglobulins and autoantibodies to nuclear antigens, it is suggested that these manifestations of liver in BTLA−/− mice correspond to type I AIH in humans.

We also evaluated the inflammatory cell infiltration of the liver semi-quantitatively by counting large and small inflammatory cell foci on HE-stained sections. As shown in Figure 3M, 75% (9 out of 12) of BTLA−/− mice showed distinct formation of inflammatory cell foci in both portal tract areas and hepatic lobules, whereas none of WT mice showed the formation of apparent foci in the liver (Figure 3M). These data indicate that BTLA−/− mice develop AIH-like disease with high penetrance at 12 months of age.

Blood chemical examinations were also performed on BTLA−/− mice at 3, 6 and 12 months of age. The levels of AST and ALT in sera were normal in BTLA−/− mice at 3 months of age (data not shown), started to increase at 6 months, and were significantly increased at 12 months (Figure 4A). The frequencies of increased release of ALT and AST were 64% (7/11) and 45% (5/11), respectively, in BTLA−/− mice at 12 months of age. On the other hand, the levels of γ–GTP, ALP and T-Bil, markers of biliary tract damage, were not significantly increased (Figure 4A).

Figure 4
Liver dysfunction and short lifespan in BTLA−/− mice

We then compared the long-term survival of BTLA−/− mice and littermate WT mice. BTLA−/− mice started to die after the age of 7 months and had a significant reduction in the survival rate thereafter as compared with WT mice (log-rank test, p<0.05) (Figure 4B). The analysis of sick BTLA−/− mice at 9 months of age revealed that these mice were severely affected with liver manifestation, suggesting that that AIH-like disease may be involved in the early death of BTLA−/− mice.

Activated CD4+ T cells and NK T cells are increased in the liver of BTLA−/− mice

To characterize the immune dysregulation causing the liver damage of BTLA−/− mice, we examined infiltrating cells in the liver of 12-month-old BTLA−/− mice. As shown in Figure 5A, the infiltrating cells in the liver of BTLA−/− mice were composed of various immune cell types. Among them, CD4+ T cells and NK T cells were significantly increased in the liver of BTLA−/− mice as compared with those in WT mice (n = 5, p<0.05). Immunohistochemistry of liver sections of BTLA−/− mice also showed that focal accumulation of mononuclear cells mainly consisted of CD4+ T cells and NK T cells (data not shown). In addition, the frequency of activated CD4+ T cells was increased in the liver of BTLA−/− mice (Figure 5B). In contrast, CD8+ T cells, NK cells, B cells, granulocytes and macrophages were not significantly increased in the liver of BTLA−/−mice. These results suggest that activated CD4+ T cells and NK T cells function as effector cells in inducing AIH-like disease in BTLA−/− mice.

Figure 5
Flow cytometric analysis of liver infiltrates in BTLA−/− mice

Interestingly, CD8+ T cells expressing IL-2 receptor β chain (CD122) (CD8+ CD122+ T cells) were significantly decreased in the liver and spleen of BTLA−/− mice as compared with those in WT mice (n = 5, p<0.05) (Figure 5C). Because CD8+ CD122+ T cells have been suggested to have an immunoregulatory function (21, 22), it is suggested that the decrease of CD8+ CD122+ T cells in the liver might be involved in the activation of CD4+ T cells and NK T cells and thus in the induction of AIH-like disease in BTLA−/− mice.

Spontaneous development of inflammatory cell infiltration in multiple organs in BTLA−/− mice

AIH is one of the representative complications of Sjögren’s syndrome (23). To explore the manifestations of Sjögren’s syndrome in BTLA−/− mice, we performed the histological examination of other organs. As shown in Figure 6A–D, the inflammatory cell infiltration that mainly consisted of lymphocytes was found in tissue sections of salivary glands of 12-month-old BTLA−/− mice. The inflammatory cell infiltration was often found as inflammatory foci around the ducts (Figure 6C, D). Inflammatory cell infiltration around the bronchus in the lung, which is the most common lung involvement in primary Sjögren’s syndrome (24, 25), was also found in BTLA−/− mice (Figure 6G, H). In addition, BTLA−/− mice showed lymphocytic infiltration in parenchymal cells in pancreas, resulting in partial destruction of the acinic epithelia (Figure 6K, L). Semi-quantitative evaluation of the sections also showed that distinct inflammatory cell foci were found only in the salivary glands, lung and pancreas of BTLA−/− mice at 12 months of age (Figure 6Q–S). Along with the fact that BTLA−/− mice show increased anti-SS-A antibody (Figure 1B), it is suggested that BTLA−/− mice exhibit the multiorgan lymphocytic infiltration by similar mechanisms operated in patients with Sjögren’s syndrome.

Figure 6
Histological analysis of various organs in BTLA−/− mice

Interestingly, although BTLA−/− mice exhibited high titer of anti-dsDNA antibody (Figure 1B), BTLA−/− mice did not show any pathological abnormalities in kidney (Figure 6O, P). Kidney sections were also stained by periodic acid methenamine silver (PAM) and periodic-acid Schiff (PAS) for further pathological examination, but we could not find any pathological changes in the kidney of BTLA−/− mice (data not shown). This is consistent with normal levels of BUN and urinary protein in BTLA−/− mice (data not shown). Moreover, 12-month-old BTLA−/− mice did not show any pathological abnormalities in heart, stomach, skin or joints.

Discussion

In this study, we show that BTLA plays an indispensable role in the maintenance of self-tolerance and thus the prevention of autoimmunity. We found that 12-month-old BTLA−/− mice but not 3-month-old BTLA−/− mice exhibited hyper-γ-globulinemia, autoantibodies to nuclear antigens and the increase in activated CD4+ T cells in the periphery (Figure 1 and and2).2). Furthermore, BTLA−/− mice spontaneously developed AIH-like disease characterized by mononuclear cell infiltration in the portal tracts and hepatic lobules associated with interface hepatitis, spotty necrosis, endothelialitis and bile duct damage (Figure 3) and by the increase in activated CD4+ T cells and NK T cells (Figure 5). Moreover, BTLA−/− mice developed inflammatory changes in the salivary glands, lungs and pancreas, which are similar to Sjögren’s syndrome (Figure 6), an autoimmune disorder commonly associated with AIH. Taken together, these results suggest that the deficiency of BTLA causes the breakdown of self-tolerance, resulting in the development of autoimmune diseases similar to AIH and Sjögren’s syndrome.

We show that BTLA plays an important role in the maintenance of tolerance to self-antigens. Accumulating evidence suggests that the inhibition of lymphocyte activation by inhibitory coreceptors is involved in the maintenance of immune homeostasis and tolerance. Involvement of CTLA-4 and PD-1 in this process has been evidenced by the analysis of mice lacking these molecules (47). In addition, we have previously shown that BTLA−/− mice exhibit increased susceptibility to experimental autoimmune encephalomyelitis (8). It has also been shown that BTLA−/− mice exhibit rapid rejection of partially MHC-mismatched cardiac allografts (26) and persistent allergic airway inflammation following antigen challenge (27). These previous studies indicate that BTLA negatively regulates experimentally induced immune responses in vivo. Our present study clearly shows that BTLA is also involved in the maintenance of tolerance to self-antigens and acts as a negative regulator for the development of autoimmune diseases. Because the expression levels of PD-1 on BTLA−/− CD4+ T cells are similar to those in WT CD4+ T cells (data not shown), it is suggested that there is no compensatory mechanism between PD-1 and BTLA and that they seem to act independently to maintain immune tolerance.

We demonstrated that BTLA−/− mice on a 129SvEv background developed inflammatory cell infiltration in the liver, lung, salivary glands and pancreas with high penetrance and a disease latency of 6–12 months (Figures 3 and and6).6). On the other hand, BTLA−/− mice did not develop inflammatory cell infiltration in the kidney, heart, stomach, skin or joints (Figure 6 and data not shown). With regard to the affected organs, it is interesting that the expression pattern of HVEM (10, 28, 29), the ligand for BTLA, largely coincides with the affected organs in BTLA−/− mice. In the case of PD-1-deficient mice, it has been demonstrated that the organs affected by the autoimmune disease depends on genetic backgrounds (5, 6). PD-1-deficient mice on a C57BL/6 background develop lupus-like glomerulonephritis and proliferative arthritis (5), whereas PD-1-deficient mice on a BALB/c background develop dilated cardiomyopathy (6). A possibility that the genetic background may influence the affected organs in BTLA−/−mice is under investigation in our laboratory.

We demonstrate that BTLA prevents autoantibody production to nuclear antigens, as indicated by the gradual increase in ANA, anti-dsDNA and anti-SS-A antibodies with age in BTLA−/− mice (Figure 1B). It has recently been shown that activation of diverse repertoires of autoreactive CD4+ T cells but not limited repertoires of CD4+ T cells is required for inducing the loss of B cell tolerance to nuclear antigens (30). It has also been shown that a greater strength of B cell receptor signaling is required for the breakdown of the tolerance of nuclear antigen-specific B cells (31). Our current study of BTLA−/− mice proves that BTLA deficiency induces not only polyclonal B cell and T cell activation (i.e. hyper-γ-globulinemia and an increase in activated T cells) but also the breakdown of B cell tolerance. Considering normal negative selection of thymocytes in BTLA−/− mice (Figure 2), the breakdown of self-tolerance seems due to the failure of peripheral tolerance.

In this regard, interestingly, we found that CD8+ CD122+ T cells, which have been suggested as a lineage of regulatory T cells (21, 22), were decreased in the liver and spleen of BTLA−/− mice (Figure 5C), suggesting that BTLA may play a role in the development and/or survival of CD8+ CD122+ T cells. In contrast, we found that CD4 + CD25+ Foxp3+ regulatory T cells were not decreased in BTLA−/− mice (Figure 2B). Because it has been demonstrated that CD8+ CD122+ T cells exhibits the inhibitory function not only on CD8+ T cells but also on CD4+ T cells (21, 22), the development of AIH-like disease and autoantibody production to nuclear antigens may be accounted for in part by the decrease of CD8+ CD122+ T cells in BTLA−/− mice. Because BTLA is expressed on B cells and antigen-presenting cells such as dendritic cells (DCs) (9, 32), it is also possible that these cells are involved in the breakdown of self-tolerance in BTLA−/−mice. These possibilities are under investigation in our laboratory.

AIH in humans is an inflammatory disorder of the liver, which is characterized by the presence of interface hepatitis on histological examination, hyper-γ-globulinemia and autoantibodies to nuclear antigens (20, 33). Infiltrating cells in the portal zone of AIH are CD4+ T cells and B cells, and portal plasma cell infiltration typifies the disorder (3336). The composition of inflammatory cell infiltration in the liver of BTLA−/− mice matched well with the case of AIH in humans. BTLA−/− mice also developed endothelialitis and inflammation of bile ducts in the liver (Figure 3). These pathological findings are often found in patients with acute transplant rejection (37) and GVHD after bone marrow transplantation (38, 39). Taken together, these results suggest that inflammation of liver in BTLA−/− mice is caused by immune dysregulation.

In addition to CD4+ T cells, we found that NK T cells were dominant inflammatory cells in AIH-like disease of BTLA−/− mice (Figure 5A). It has been demonstrated that NK T cells are responsible for the development of Concanavalin A (Con A)-induced hepatitis (40, 41), which is considered to be a mouse model of AIH. We found that mouse NK T cells constitutively express BTLA (data not shown). It has also been demonstrated that HVEM-deficient mice exhibit increased morbidity and mortality in Con A-induced hepatitis as compared with WT mice (42). Taken together, it is suggested that the deficiency of BTLA/HVEM interaction causes the activation of NK T cells in the liver and induces AIH-like disease in BTLA−/− mice. The initial intrinsic stimuli for NK T cells in the liver of BTLA−/− mice remain to be elucidated.

In conclusion, we have demonstrated that the deficiency of BTLA causes the breakdown of self-tolerance, resulting in the development of AIH-like disease and lymphocytic infiltration in multiple organs. These results indicate that BTLA plays a critical role in the maintenance of self-tolerance and the prevention of autoimmunity and suggest that the enhancement of BTLA signaling by agonistic ligands might be useful for the treatment of autoimmune diseases.

Acknowledgments

This work was supported in part by Grants-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government.

Dedicated to Hiroshi Ishikura, a pioneer in liver pathology.

We thank Ms. Nagashima for animal care.

Abbreviations

BTLA
B and T lymphocyte attenuator
HVEM
herpesvirus entry mediator

Footnotes

All the authors have no financial conflict of interest.

References

1. Sharpe AH, Freeman GJ. The B7-CD28 superfamily. Nat Rev Immunol. 2002;2:116–26. [PubMed]
2. Leibson PJ. The regulation of lymphocyte activation by inhibitory receptors. Curr Opin Immunol. 2004;16:328–36. [PubMed]
3. Chen L. Co-inhibitory molecules of the B7-CD28 family in the control of T-cell immunity. Nat Rev Immunol. 2004;4:336–47. [PubMed]
4. Tivol EA, Borriello F, Schweitzer AN, Lynch WP, Bluestone JA, Sharpe AH. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity. 1995;3:541–7. [PubMed]
5. Nishimura H, Nose M, Hiai H, Minato N, Honjo T. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity. 1999;11:141–51. [PubMed]
6. Nishimura H, Okazaki T, Tanaka Y, Nakatani K, Hara M, Matsumori A, et al. Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science. 2001;291:319–22. [PubMed]
7. Okazaki T, Tanaka Y, Nishio R, Mitsuiye T, Mizoguchi A, Wang J, et al. Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-1-deficient mice. Nat Med. 2003;9:1477–83. [PubMed]
8. Watanabe N, Gavrieli M, Sedy JR, Yang J, Fallarino F, Loftin SK, et al. BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1. Nat Immunol. 2003;4:670–9. [PubMed]
9. Han P, Goularte OD, Rufner K, Wilkinson B, Kaye J. An inhibitory Ig superfamily protein expressed by lymphocytes and APCs is also an early marker of thymocyte positive selection. J Immunol. 2004;172:5931–9. [PubMed]
10. Montgomery RI, Warner MS, Lum BJ, Spear PG. Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family. Cell. 1996;87:427–36. [PubMed]
11. Sedy JR, Gavrieli M, Potter KG, Hurchla MA, Lindsley RC, Hildner K, et al. B and T lymphocyte attenuator regulates T cell activation through interaction with herpesvirus entry mediator. Nat Immunol. 2005;6:90–8. [PubMed]
12. Gonzalez LC, Loyet KM, Calemine-Fenaux J, Chauhan V, Wranik B, Ouyang W, et al. A coreceptor interaction between the CD28 and TNF receptor family members B and T lymphocyte attenuator and herpesvirus entry mediator. Proc Natl Acad Sci U S A. 2005;102:1116–21. [PMC free article] [PubMed]
13. Gavrieli M, Watanabe N, Loftin SK, Murphy TL, Murphy KM. Characterization of phosphotyrosine binding motifs in the cytoplasmic domain of B and T lymphocyte attenuator required for association with protein tyrosine phosphatases SHP-1 and SHP-2. Biochem Biophys Res Commun. 2003;312:1236–43. [PubMed]
14. Williams LM, Rudensky AY. Maintenance of the Foxp3-dependent developmental program in mature regulatory T cells requires continued expression of Foxp3. Nat Immunol. 2007;8:277–84. [PubMed]
15. Malhi H, Annamaneni P, Slehria S, Joseph B, Bhargava KK, Palestro CJ, et al. Cyclophosphamide disrupts hepatic sinusoidal endothelium and improves transplanted cell engraftment in rat liver. Hepatology. 2002;36:112–21. [PubMed]
16. Matsumoto K, Watanabe N, Akikusa B, Kurasawa K, Matsumura R, Saito Y, et al. Fc receptor-independent development of autoimmune glomerulonephritis in lupus-prone MRL/lpr mice. Arthritis Rheum. 2003;48:486–94. [PubMed]
17. Kappler JW, Staerz U, White J, Marrack PC. Self-tolerance eliminates T cells specific for Mls-modified products of the major histocompatibility complex. Nature. 1988;332:35–40. [PubMed]
18. MacDonald HR, Schneider R, Lees RK, Howe RC, Acha-Orbea H, Festenstein H, et al. T-cell receptor Vβ use predicts reactivity and tolerance to Mlsa-encoded antigens. Nature. 1988;332:40–5. [PubMed]
19. Yoshida R, Yoshioka T, Yamane S, Matsutani T, Toyosaki-Maeda T, Tsuruta Y, et al. A new method for quantitative analysis of the mouse T-cell receptor V region repertoires: comparison of repertoires among strains. Immunogenetics. 2000;52:35–45. [PubMed]
20. Czaja AJ, Freese DK. Diagnosis and treatment of autoimmune hepatitis. Hepatology. 2002;36:479–97. [PubMed]
21. Rifa’i M, Kawamoto Y, Nakashima I, Suzuki H. Essential roles of CD8+CD122+ regulatory T cells in the maintenance of T cell homeostasis. J Exp Med. 2004;200:1123–34. [PMC free article] [PubMed]
22. Endharti AT, Rifa’i M, Shi Z, Fukuoka Y, Nakahara Y, Kawamoto Y, et al. Cutting edge: CD8+CD122+ regulatory T cells produce IL-10 to suppress IFN-γ production and proliferation of CD8+ T cells. J Immunol. 2005;175:7093–7. [PubMed]
23. Lindgren S, Manthorpe R, Eriksson S. Autoimmune liver disease in patients with primary Sjogren’s syndrome. J Hepatol. 1994;20:354–8. [PubMed]
24. Constantopoulos SH, Papadimitriou CS, Moutsopoulos HM. Respiratory manifestations in primary Sjogren’s syndrome. A clinical, functional, and histologic study. Chest. 1985;88:226–9. [PubMed]
25. Franquet T, Gimenez A, Monill JM, Diaz C, Geli C. Primary Sjogren’s syndrome and associated lung disease: CT findings in 50 patients. AJR Am J Roentgenol. 1997;169:655–8. [PubMed]
26. Tao R, Wang L, Han R, Wang T, Ye Q, Honjo T, et al. Differential effects of B and T lymphocyte attenuator and programmed death-1 on acceptance of partially versus fully MHC-mismatched cardiac allografts. J Immunol. 2005;175:5774–82. [PubMed]
27. Deppong C, Juehne TI, Hurchla M, Friend LD, Shah DD, Rose CM, et al. Cutting edge: B and T lymphocyte attenuator and programmed death receptor-1 inhibitory receptors are required for termination of acute allergic airway inflammation. J Immunol. 2006;176:3909–13. [PubMed]
28. Marsters SA, Ayres TM, Skubatch M, Gray CL, Rothe M, Ashkenazi A. Herpesvirus entry mediator, a member of the tumor necrosis factor receptor (TNFR) family, interacts with members of the TNFR-associated factor family and activates the transcription factors NF-kappaB and AP-1. J Biol Chem. 1997;272:14029–32. [PubMed]
29. Hsu H, Solovyev I, Colombero A, Elliott R, Kelley M, Boyle WJ. ATAR, a novel tumor necrosis factor receptor family member, signals through TRAF2 and TRAF5. J Biol Chem. 1997;272:13471–4. [PubMed]
30. Busser BW, Adair BS, Erikson J, Laufer TM. Activation of diverse repertoires of autoreactive T cells enhances the loss of anti-dsDNA B cell tolerance. J Clin Invest. 2003;112:1361–71. [PMC free article] [PubMed]
31. Qian Y, Santiago C, Borrero M, Tedder TF, Clarke SH. Lupus-specific antiribonucleoprotein B cell tolerance in nonautoimmune mice is maintained by differentiation to B-1 and governed by B cell receptor signaling thresholds. J Immunol. 2001;166:2412–9. [PubMed]
32. Hurchla MA, Sedy JR, Gavrieli M, Drake CG, Murphy TL, Murphy KM. B and T lymphocyte attenuator exhibits structural and expression polymorphisms and is highly Induced in anergic CD4+ T cells. J Immunol. 2005;174:3377–85. [PubMed]
33. Alvarez F, Berg PA, Bianchi FB, Bianchi L, Burroughs AK, Cancado EL, et al. International Autoimmune Hepatitis Group Report: review of criteria for diagnosis of autoimmune hepatitis. J Hepatol. 1999;31:929–38. [PubMed]
34. Czaja AJ, Carpenter HA. Sensitivity, specificity, and predictability of biopsy interpretations in chronic hepatitis. Gastroenterology. 1993;105:1824–32. [PubMed]
35. Johnson PJ, McFarlane IG. Meeting report: International Autoimmune Hepatitis Group. Hepatology. 1993;18:998–1005. [PubMed]
36. Frazer IH, Mackay IR, Bell J, Becker G. The cellular infiltrate in the liver in auto-immune chronic active hepatitis: analysis with monoclonal antibodies. Liver. 1985;5:162–72. [PubMed]
37. Snover DC, Sibley RK, Freese DK, Sharp HL, Bloomer JR, Najarian JS, et al. Orthotopic liver transplantation: a pathological study of 63 serial liver biopsies from 17 patients with special reference to the diagnostic features and natural history of rejection. Hepatology. 1984;4:1212–22. [PubMed]
38. Scheuer PJ, Lefkowitch JH. Liver Biopsy Interpretation. 6. London: W.B. Saunders; 2000.
39. McDonald GB, Shulman HM, Sullivan KM, Spencer GD. Intestinal and hepatic complications of human bone marrow transplantation. Part I. Gastroenterology. 1986;90:460–77. [PubMed]
40. Kaneko Y, Harada M, Kawano T, Yamashita M, Shibata Y, Gejyo F, et al. Augmentation of Vα14 NKT cell-mediated cytotoxicity by interleukin 4 in an autocrine mechanism resulting in the development of concanavalin A-induced hepatitis. J Exp Med. 2000;191:105–14. [PMC free article] [PubMed]
41. Takeda K, Hayakawa Y, Van Kaer L, Matsuda H, Yagita H, Okumura K. Critical contribution of liver natural killer T cells to a murine model of hepatitis. Proc Natl Acad Sci U S A. 2000;97:5498–503. [PMC free article] [PubMed]
42. Wang Y, Subudhi SK, Anders RA, Lo J, Sun Y, Blink S, et al. The role of herpesvirus entry mediator as a negative regulator of T cell-mediated responses. J Clin Invest. 2005;115:711–7. [PMC free article] [PubMed]
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