Inadequate induction of suppressor of cytokine signaling-1 causes systemic autoimmune diseases
Minoru Fujimoto1,
Hiroko Tsutsui2,5,
Ouyang Xinshou1,
Masanori Tokumoto3,
Dai Watanabe1,
Yoshihito Shima1,
Tomohiro Yoshimoto2,5,
Hideki Hirakata3,
Ichiro Kawase1,
Kenji Nakanishi2,5,
Tadamitsu Kishimoto4 and
Tetsuji Naka1
1 Department of Molecular Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita City, Osaka 565-0871, Japan 2 Department of Immunology and Medical Zoology, Hyogo College of Medicine, Nishinomiya 663-8501, Japan 3 Department of Medicine and Clinical Science, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan 4 Osaka University, Osaka 565-0871, Japan 5 Core Research for Evolutional Science and Technology, Japan Science and Technology Corp., Tokyo 101-0062, Japan
Correspondence to: T. Naka; E-mail: naka{at}imed3.med.osaka-u.ac.jp
Transmitting editor: T. Watanabe
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Abstract
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Suppressor of cytokine signaling (SOCS)-1 is a cytokine-inducible, negative regulatory molecule of Janus kinases (JAK) and its deficiency causes hyper-response to various cytokines. SOCS-1/ mice spontaneously develop a fatal disease depending on aberrantly activated lymphocytes. Here, we show that partial restoration of SOCS-1 in lymphoid cells rescues SOCS-1/ mice from the early-onset fatal disease, indicating that SOCS-1 expression in vivo is especially required in lymphocytes. However, SOCS-1 expression in these SOCS-1-restored mutant mice (Eµ-SOCS-1/ mice) was insufficient for proper down-regulation of its target signaling, and these mice spontaneously exhibit hyperactivation of lymphocytes, an increase in the levels of serum Ig and anti-DNA autoantibodies, and glomerulonephritis with glomerular IgG deposition. These phenotypes resemble those of murine systemic autoimmune diseases, models for systemic lupus erythematosus (SLE). Interestingly, similar phenotypes were also observed in adult female SOCS-1+/ mice, indicating that the autoimmune phenotypes of these mice can be ascribed primarily to the inadequate expression of SOCS-1. In addition, autoimmune phenotypes were not observed in SOCS-1+/CD4/ mice, suggesting that autoimmunity is dependent on hyper-activated CD4+ T cells. Our findings also suggest that insufficient expression of SOCS-1 results in impaired function of CD25+CD4+ regulatory T cells, which may contribute to aberrant activation of CD4+ T cells. These findings suggest that dysfunction of SOCS-1 can be a pathogenic factor of systemic autoimmune diseases such as SLE.
Keywords: autoantibody, Janus kinase, STAT-induced STAT inhibitor, suppressor of cytokine signaling, systemic lupus erythematosus
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Introduction
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Systemic autoimmune diseases are believed to emerge from a wide range of abnormalities in immunoregulation. The most representative systemic autoimmune disease in humans is systemic lupus erythematosus (SLE), which is characterized by the presence of activated T cells and B cells in conjunction with the development of autoantibodies, particularly those against dsDNA, and by the involvement of multiple tissue injuries including skin and kidney. The etiology of SLE involves both genetic and environmental factors, although the details are largely unknown (1,2). However, various cytokines, such as IL-4 and IFN, have been reported to participate in the pathogenesis of systemic autoimmune disease via induction of excessive production of Ig, including autoantibodies (37).
Suppressor of cytokine signaling (SOCS)-1 is a cytokine-inducible intracellular molecule. SOCS-1 interacts with phosphorylated Janus kinases (JAK), and functions as a negative feedback factor for signaling of cytokines such as IFN-
, IL-4 and IL-2 (810). Absence of SOCS-1 results in excessive responsiveness to these cytokines. SOCS-1-deficient (SOCS-1/) mice die within 3 weeks after birth of a complex disease, such as fatal liver injuries characterized by fatty degeneration and necrosis, severe lymphocytopenia, and dense infiltration of markedly activated lymphocytes into multiple organs (914). RAG2/ mice reconstituted with SOCS-1/ lymphocytes develop similar diseases, whereas SOCS-1/RAG double-knockout mice that lack mature lymphocytes are rescued from the disease (11,15,16). In addition, T cells in SOCS-1/ mice spontaneously exhibit an activated phenotype (11,15). These results indicate that these pathological changes are caused by SOCS-1/ lymphocytes and SOCS-1 is essential for protection against the harmful activation of T cells, presumably via inhibition of excessive responsiveness to cytokines.
It is of interest to investigate whether the correction of SOCS-1 deficiency only in lymphocytes can rescue SOCS-1/ mice from the lymphocyte-mediated multiple tissue injuries. In this study, we crossed Eµ-SOCS-1 transgenic (Tg) mice with SOCS-1/ mice to generate Eµ-SOCS-1/ mice that express a certain level of SOCS-1 selectively in lymphoid cells of SOCS-1/ background mice. Although transgene-mediated expression of SOCS-1 prevented perinatal lethality of SOCS-1/ mice, it achieved only partial correction of SOCS-1 in lymphocytes and failed to prevent abnormal lymphocyte activation. In addition, Eµ-SOCS-1/ mice manifested other pathological changes reminiscent of murine systemic autoimmune diseases. More interestingly, this was also the case for some adult female SOCS-1+/ mice, but not in SOCS-1+/ CD4/ mice. Our findings also imply that insufficient SOCS-1 induces the dysfunction of CD25+CD4+ regulatory T cells, subpopulations of CD4+ T cells critically involved in the inhibition of harmful immunopathological responses. These findings suggest that proper expression of SOCS-1 is essential for the immune homeostasis as well as the prevention of systemic autoimmunity.
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Methods
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Generation of Eµ-SOCS-1 Tg mice
SOCS-1 cDNA was inserted into the XhoI site of the pEµIgH vector containing the human Ig enhancer (hEµ) and murine IgH promoter (17). This vector was linearized and injected into fertilized eggs of C57BL/6 mice. Founder mice and their offspring were screened by PCR using tail DNA for the establishment of several Tg lines. In this study, we used Tg mice obtained from a line named B-8.
SOCS-1/ and SOCS-1+/ mice
SOCS-1/ mice on a C57BL/6 background were reported previously (15). To obtain Eµ-SOCS-1/ mice, Eµ-SOCS-1 Tg mice were crossed with C57BL/6 SOCS-1+/ mice and the offspring Eµ-SOCS-1+/ mice were further crossed with SOCS-1+/ mice. CD4/ mice (129 x C57BL/6) were obtained from Jackson Laboratory (Bar Harbor, ME) and maintained on a C57BL/6 background. C57BL/6 SOCS-1+/ mice were crossed with CD4/ mice to obtain SOCS-1+/ CD4/ mice.
Flow cytometry analysis
Single-cell suspensions were prepared as reported previously (18). Cells were stained with FITC-, phycoerythrin-, PerCP- or allophycocyanin-conjugated antibodies against CD4, CD8, CD25, CD40, CD62L, CD80, CD86, I-Ab and B220 (all purchased from PharMingen, San Diego, CA), and analyzed on a FACSCalibur using CellQuest software (Becton Dickinson, San Jose, CA).
Western blot analysis
To examine the expression of SOCS-1 protein, small pieces of indicated tissues were homogenized with pellet mixer in ice-cold lysis buffer and supernatants were used as cell lysates. To examine IFN-
-induced STAT1 activation or IL-2-induced STAT5 activation, lymph node cells or sorted CD25+ CD4+ T cells were stimulated with 50 ng/ml of IFN-
(PeproTech, London, UK) or 500 U/ml of IL-2 (R & D Systems, Minneapolis, MN) respectively for the indicated minutes and lysed in ice-cold buffer. Cell lysates were subjected to SDSPAGE as described previously (18). Samples transferred onto a filter were treated with the following antibodies: anti-JAB/SOCS-1 (IBL, Fujioka, Japan), anti-phospho-STAT1 (Upstate Biotech nology, Lake Placid, NY), anti-STAT1 (Transduction Laboratory, Lexington, KY), anti-phospho-STAT5 (Cell Signal ing Technology, Beverly, MA) or anti-STAT5 (Santa Cruz Biotechnology, Santa Cruz, CA) and visualized as described previously (18).
RT-PCR
RT-PCR analysis of SOCS-1 and G3PDH expression was performed essentially as described previously (19).
Histological analysis
Formalin-fixed tissues were stained with hematoxylin & eosin or periodic acidSchiff. For detection of IgG deposits in kidneys, kidneys were rapidly frozen in liquid nitrogen and 2-µm thick cryostat sections were fixed in 100% acetone for 15 min. The sections were incubated with FITC-conjugated goat anti-mouse IgG (ICN Biomedicals, Irvine, CA) at a concentration of 10 µg/ml, overnight at 4°C.
Isolation of dendritic cells (DC)
To obtain DC, single-cell suspensions were prepared from collagenase-injected spleens of Eµ-SOCS-1/ and Eµ-SOCS-1+/+ mice (H-2b) (20). After being incubated in a plastic dish with 10-cm diameter for 2 h, non-adherent cells were removed and adherent cells were cultured further overnight. Spontaneously detached cells were then collected, and CD11c+ cells isolated by the usage of anti-CD11c-conjugated magnetic beads and autoMACS (Miltenyi Biotec, Auburn, CA) were used as DC.
Serum Ig
Serum Ig levels were determined by ELISA according to the manufacturers instructions. Antibodies used were alkaline phosphatase-conjugated anti-IgG1 and anti-IgG1 (Southern Biotechnology, Birmingham, AL), biotinylated anti-IgG2a and anti-IgG2a (PharMingen), biotinylated anti-IgE and anti-IgE, and streptavidin-conjugated horseradish peroxidase (PharMingen).
Detection of anti-dsDNA and anti-ssDNA antibodies
Serum levels of anti-dsDNA or anti-ssDNA antibodies were measured with commercially available ELISA kits (Shibayagi, Shibukawa, Japan) according to the manufacturers instructions.
Proliferation assay for thymocytes
Thymocytes (2 x 105/well) were cultured in 96-well plates as described previously (18) in the presence of suboptimal dose of phorbol myristate acetate (10 ng/ml; Nakarai Tesque, Kyoto, Japan) and indicated doses of IL-2 (PeproTech). Thymocyte proliferation was determined by the usage of Cell Counting Kit-8 (Dojin, Kumamoto, Japan) as described previously (18).
B cell proliferation assay
Resting B cells were prepared from SOCS-1+/ or wild-type mice according to the method described previously (21). B cells were incubated with 10 µg/ml of anti-IgM in the presence of various doses of rIL-4 (R & D Systems) for 3 days and pulsed with [3H]thymidine for the last 16 h.
Northern blot analysis
Thymocytes or splenocytes from Eµ-SOCS-1 Tg mice and their non-Tg littermates were incubated with FITC-conjugated anti-CD3e antibodies (PharMingen) or a mixture of FITC-conjugated antibodies against CD19 (PharMingen) and MHC class II (eBiosience, San Diego, CA) respectively. These cells were then treated with anti-FITC beads (Miltenyi Biotec) and subjected to MACS systems to obtain both positive and negative fractions (CD3+/CD3 for thymocytes and T/non-T fractions for splenocytes). Where indicated, splenic non-Tg T cells were stimulated with IFN-
(PeproTech) for 30 min. Spleen and lymph node cells from wild-type mice were incubated with FITC-conjugated anti-CD25 antibodies (eBioscience) and then with anti-FITC beads (Miltenyi Biotec). Positively selected cells by MACS systems (Miltenyi Biotec) were used as CD25+CD4+ regulatory T cells. Residual cells were further labeled with a mixture of FITC-conjugated antibodies including anti-CD8, anti-CD19, anti-CD25 (PharMingen) and anti-MHC class II (eBioscience), and then with anti-FITC beads, and negatively selected cells were used as CD25CD4+ T cells. CD25CD4+ T cells were further stimulated for 1 h with IFN-
or IL-4 (100 ng/ml; PeproTech) or left untreated. Using RNA-Bee (Tel-Test, Friendswood, TX), total RNA was obtained from the cells described above. RNA (2 µg/sample) was subjected to agarose gel electrophoresis and northern analysis was performed using the DIG Northern Starter Kit (Roche Diagnostics, Manheim, Germany) according to the manufacturers instructions. SOCS-1 cDNA subcloned into pBluescript II SK+ was used to prepare the riboprobe.
Isolation of CD25+CD4+ T cells and proliferation assay
Spleen cells were incubated with a mixture of anti-CD8, anti-CD16/anti-CD32, anti-I-Ab and anti-B220 magnetic microbeads (Miltenyi Biotec), and CD4+ cells were negatively selected by autoMACS. CD25+ cells were positively selected from the cell suspension following incubation with anti-CD4 microbeads (Miltenyi Biotec) and were used as CD25+CD4+ regulatory T cells. Residual CD25 cells were used as responder cells. Irradiated spleen cells (30 Gy) were used as antigen-presenting cells (APC). Various numbers of CD25+CD4+ regulatory T cells, CD25CD4+ cells (1 x 105/well) and irradiated APC (2 x 105/well) were incubated with 0.5 µg/ml of soluble anti-CD3 (PharMingen). Where indicated, IL-2 was also added in culture media. On day 3, after 16-h pulsing with 1 µCi [3H]thymidine/well (Amersham Pharmacia Biotech), cultures were harvested and radionuclide uptake was measured by scintillation counting.
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Results
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Partial recovery of SOCS-1 in lymphocytes prevents early death of SOCS-1/ mice, but leads to generation of various inflammatory diseases
Previous analyses have shown that SOCS-1 is preferentially expressed in lymphoid organs, especially in thymus, and is a critical regulator of lymphocyte activation. To determine whether correction of SOCS-1 deficiency only in lymphocytes can rescue SOCS-1/ mice from lymphocyte-mediated fatal tissue injuries, we first generated SOCS-1 Tg mice (Eµ-SOCS-1 Tg mice) that express SOCS-1 in lymphoid cells under the control of the human Ig enhancer (Eµ) and murine IgH promoter, and crossed them with SOCS-1/ mice. As shown in Fig. 1(a), remarkable amounts of SOCS-1 protein were detected only in thymus of Eµ-SOCS-1 Tg mice. By RT-PCR analysis, transgene-derived SOCS-1 was also detected with lesser amounts in bone marrow (data not shown), spleen and liver of Eµ-SOCS-1 Tg mice (Fig. 1b), which suggests that the transgene expression is especially active in thymocytes, but less efficient in peripheral lymphocytes. Indeed, remarkable levels of transgene-derived SOCS-1 mRNA were observed both in immature CD3 thymocytes and mature CD3+ thymocytes of Eµ-SOCS-1 Tg mice, while reduced levels were seen in splenic T cells and non-T cells, the majority of which were B cells (Fig. 1c). As can be seen in Fig. 1(c), SOCS-1 expression in splenic lymphocytes of Eµ-SOCS-1 Tg mice slightly exceeded the basal SOCS-1 expression in those of wild-type mice, but did not reach to the expression level of wild-type thymocytes and IFN-
-stimulated T cells.
Eµ-SOCS-1 Tg mice appeared healthy and exhibited no gross abnormalities under specific pathogen-free conditions (Fig. 1e), although a mild decrease in the number of lymphocytes was observed (data not shown) as in the case of another lymphocyte-specific SOCS-1 Tg mice (18). We next generated Eµ-SOCS-1 Tg mice lacking endogenous SOCS-1 (Eµ-SOCS-1/ mice) by crossing Eµ-SOCS-1 Tg mice (referred to hereafter as Eµ-SOCS-1+/+ mice) with SOCS-1/ mice and re-introduced the expression of SOCS-1 specifically in lymphocytes of SOCS-1/ background mice. We examined whether the introduced SOCS-1 in peripheral lymphocytes can normally suppress cytokine signaling. When lymph node cells from Eµ-SOCS-1/ mice were stimulated with IFN-
, a potent inducer of SOCS-1, STAT1 activation was clearly detectable as in the case of wild-type cells (data not shown), but obviously prolonged as compared to wild-type (wild-type) cells (data not shown) and Eµ-SOCS-1+/+ cells (Fig. 1d). This result appears to be in line with the weak expression of SOCS-1 in the periphery of Eµ-SOCS-1+/+ mice (Fig.1c) and suggests that SOCS-1 expression driven by the Eµ/IgH promoter is insufficient for normal down-regulation of cytokine signaling that would be achieved by the activation of the endogenous SOCS-1 promoter. These results indicate that mature lymphocytes of Eµ-SOCS-1/ mice express only a limited level of SOCS-1 that allows excessive activation of STAT.
Despite the inadequate action of SOCS-1 (Fig. 1d), most Eµ-SOCS-1/ mice grew larger than SOCS-1/ mice (Fig. 1e) and survived >3 weeks (Figs 1f and 2a). Moreover, Eµ-SOCS-1/ mice evaded the fatal hepatitis observed in SOCS-1/ mice (Fig. 2b), suggesting the importance of SOCS-1 in down-regulation of liver lymphocyte activation (15). Thus, transgene-derived expression of SOCS-1 in lymphocytes partially improved the fatal organ pathologies in SOCS-1/ mice and prevented their early death. Although they showed improvements in the structural development of immune organs compared with the parental SOCS-1/ mice (data not shown), Eµ-SOCS-1/ mice manifested pathological changes in lung and heart with perivascular infiltrates, and developed periportal infiltrates in the liver (Fig. 2b). They also developed cutaneous alterations accompanied by hair loss, hair hypopigmentation, eczema and small ulcers with moderate infiltrates (Fig. 2a and b). These pathological alterations in Eµ-SOCS-1/ mice might be the natural consequence that would occur in parental SOCS-1/ mice if they could survive much longer. Alternatively, these changes might be generated newly as a result of incomplete recovery of SOCS-1 in lymphocytes.

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Fig. 2. Gross appearance and histological analysis of Eµ-SOCS-1/ mice. (a) Appearance of adult Eµ-SOCS-1/ mice. Eµ-SOCS-1/ mice were slightly smaller than control mice, and had skin lesions such as hair hypopigmentation, alopecia (head) and eczema (tail). (b) Mononuclear cell infiltrations in various organs of Eµ-SOCS-1/ mice. Hematoxylin & eosin staining of liver, skin, lung and heart was shown for Eµ-SOCS-1/ mice and Eµ-SOCS-1+/+ mice (4 months of age). (c and d) Enhanced activation of T and B cells in Eµ-SOCS-1/ mice. Activation status of T cells (c) and B cells (d) was evaluated with flow cytometry by the expression of CD62L and CD86 respectively. (e) Up-regulation of co-stimulatory molecules on DC in Eµ-SOCS-1/ mice. Surface expression of the indicated co-stimulatory molecules on CD11c+ cells was analyzed by flow cytometry.
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Eµ-SOCS-1/ mice exhibit spontaneous activation of T cells, B cells and DC
We next investigated whether ectopic expression of SOCS-1 in lymphocytes improves spontaneous activation of various types of SOCS-1/ immune cells (11,15). Eµ-SOCS-1/ mice still disclosed significant increases in the proportion of activated T cells (Fig. 2c) and of B cells expressing a co-stimulatory molecule, CD86 (Fig. 2d). In addition, DC of Eµ-SOCS-1/ mice expressed slightly higher levels of co-stimulatory molecules (Fig. 2e). These results suggest that insufficient or null SOCS-1 may lead to spontaneous activation of various types of immune cells, which in turn may participate in the pathogenesis of multiple organ diseases in Eµ-SOCS-1/mice.
Eµ-SOCS-1/ mice develop lupus-like systemic autoimmunity
As both CD4+ T cells and B cells were abnormally activated, we measured serum levels of Ig. Both IgG2a, an Ig isotype associated with a Th1 response, and IgG1/IgE, products of a Th2 response, were noticeably elevated in Eµ-SOCS-1/ mice (Fig. 3a). This result could be attributed to the close interaction between abnormally activated effector CD4+ T cells and B cells in Eµ-SOCS-1/ mice, because germinal center (GC) formations were seen in lymph nodes and spleen of adult Eµ-SOCS-1/ mice without exogenous stimuli (data not shown).

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Fig. 3. Lupus-like abnormalities in Eµ-SOCS-1/ mice. (a) Increased Ig levels in Eµ-SOCS-1/ mice. Serum Ig levels were determined by ELISA. (b) Enhanced autoantibody production in Eµ-SOCS-1/ mice. The levels of IgG antibodies against dsDNA and ssDNA in sera from SOCS-1+/+ mice (n = 3), Eµ-SOCS-1+/+ mice (n = 8) and Eµ-SOCS-1/ mice (n = 7) were measured by ELISA (*P < 0.05 compared to other groups by the MannWhitney test). (c) Glomerulonephritis in Eµ-SOCS-1/ mice. (Top) Increased matrix and mesangial proliferation was observed in glomeruli of Eµ-SOCS-1/ mice (periodic acidSchiff staining). (Bottom) Kidney sections stained with FITC-labeled anti-mouse IgG revealed IgG deposition in glomeruli of Eµ-SOCS-1/ mice.
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These features in Eµ-SOCS-1/ mice, such as multiple inflammatory lesions, spontaneous lymphocyte activation and elevated production of Ig, are reminiscent of murine lupus models (1). We therefore examined whether Eµ-SOCS-1/ mice produce autoantibodies against DNA. Significantly, large amounts of anti-ssDNA and anti-dsDNA were detected in the sera of Eµ-SOCS-1/ mice (Fig. 3b). Examination for pathological changes in kidney, a representative organ that is injured by deposition of immune complexes in SLE, revealed mesangial cell proliferation in the glomeruli of Eµ-SOCS-1/ mice (Fig. 3c). Moreover, large amounts of IgG were found deposited on the glomeruli of Eµ-SOCS-1/ mice (Fig. 3c). These results suggest that autoantibodies including anti-DNA in Eµ-SOCS-1/ mice are sufficiently pathogenic to cause glomerulonephritis. All of these results suggest that Eµ-SOCS-1/ mice intrinsically exhibit systemic autoimmunity.
Systemic autoimmunity in SOCS-1 mutant mice is attributed to insufficient actions of SOCS-1
The absence of autoimmune phenotypes in Eµ-SOCS-1+/+ mice suggests that autoimmunity in Eµ-SOCS-1/ mice can be ascribed to the lack of adequate SOCS-1 expression. However, it is important to exclude the possibility that the Eµ transgene mainly contributes to the autoimmunity in Eµ-SOCS-1/ mice.
Recently, we and others reported a phenotype of SOCS-1+/ mice due to the defect of one SOCS-1 allele (2224). Indeed, SOCS-1+/ naive CD4+ T cells exhibited enhanced immune response, as indicated by hyper-polarization to Th1 and Th2 cells upon stimulation with IL-12 and IL-4 respectively (22). Therefore, we next screened the sera of adult (>6 months old) SOCS-1+/ mice to examine whether they also suffer from systemic autoimmunity.
Expectedly, remarkable elevation of anti-ssDNA was detected in some SOCS-1+/ mice (Fig. 4a). In particular, a significant percentage of female SOCS-1+/ mice (eight out of 23) produced increased amounts of this autoantibody compared with female SOCS-1+/+ mice (Fig. 4a; P < 0.05 by Fishers exact test). Moreover, these female SOCS-1+/ mice with high levels of anti-ssDNA antibody typically possessed increased levels of anti-dsDNA antibody, contained increased proportions of activated T cells (data not shown), and manifested inflammatory changes in lung (data not shown), liver, salivary glands and kidneys (Fig. 4b), and renal pathological alterations similar to those in Eµ-SOCS-1/ mice (Fig. 4b and c). This is reminiscent of the female gender-biased incidence in human SLE. Thus, the analysis of SOCS-1+/ mice suggests again that appropriate induction of SOCS-1 is critical for the prevention of systemic autoimmune disease. This analysis also suggests that a lack of one SOCS-1 allele is sufficient to enhance autoimmunity, especially in females.

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Fig. 4. Systemic autoimmunity in adult SOCS-1+/ mice. (a) Elevated serum anti-ssDNA antibodies in adult SOCS-1+/ mice, but not in SOCS-1+/CD4/ mice. Serum autoantibodies in SOCS-1+/+ mice (male n = 3, female n = 12), SOCS-1+/ mice (male n = 12, female n = 23) and CD4/ SOCS-1+/ mice (male n = 7, female n = 8) were measured by ELISA. Arrow indicates the titer 2 SD above the average titer of female SOCS-1+/+ controls. (b) Pathologic changes in organs of SOCS-1+/ mice. Infiltrates of mononuclear cells were present in the liver, salivary gland and kidney of SOCS-1+/ mice (hematoxylin & eosin staining). Hypercellular glomeruli were observed in the kidney of SOCS-1+/ mice (periodic acidSchiff staining; bottom). (c) IgG deposits in glomeruli of SOCS-1+/ mice. Kidneys of SOCS-1+/+ and SOCS1+/ mice were stained as described in Fig. 3(c). (d) Enhanced proliferation of SOCS-1+/ thymocytes in response to cytokines. Thymocytes from SOCS-1+/+ and SOCS-1+/ mice were stimulated with phorbol myristate acetate (10 ng/ml) with indicated doses of IL-2 for 3 days. The proliferation of thymocytes was determined by a cell counting kit. (e) Normal proliferation of SOCS-1+/ B cells. Sorted B cells from SOCS-1+/+ or SOCS-1+/ mice were stimulated with indicated doses of IL-4 with or without anti-µ. Proliferative response was determined by [3H]thymidine incorporation. (f) GC formation in lymph node of SOCS-1+/ mice. GC in lymph nodes of SOCS-1+/ mice were visualized by immunohistochemical staining with anti-PCNA antibody.
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SOCS-1 is involved in the maintenance of the proper functions of CD4+ T cells
Previous analyses of murine lupus models indicated that systemic autoimmunity is critically dependent on T cells, especially on CD4+ T cells (2). This may also be the case for SOCS-1 mutant mice because increased proportions of CD4+ T cells in Eµ-SOCS-1/ mice and diseased SOCS-1+/ mice showed an activated phenotype (Fig. 2c and data not shown). In addition, accumulated studies have indicated that aberrantly activated SOCS-1/ T cells are central to the pathology of SOCS-1/ mice (1115) and even SOCS-1+/ T cells upon stimulation showed enhanced immune responses (22). Therefore, we next focused on lymphocytes from young pre-onset SOCS-1+/ mice, because those from Eµ-SOCS-1/ mice might already be primed in vivo (Fig. 2c). We first examined the proliferation of SOCS-1+/ thymocytes in response to IL-2, a classical mitogen for T cells. As shown in Fig. 4(d), T cells from SOCS-1+/ mice showed enhanced proliferation in response to IL-2, suggesting that SOCS-1 negatively regulates IL-2-induced proliferation of T cells. In contrast, SOCS-1+/ B cells comparably proliferated in response to stimulation with anti-IgM plus IL-4 as SOCS-1+/+ cells, implying that SOCS-1 plays a minor regulatory role in the proliferation of B cells upon stimulation (Fig. 4e). Moreover, likewise in Eµ-SOCS-1/ mice, we could observe GC formations without exogenous stimuli in lymph nodes and spleen of diseased female SOCS-1+/ mice (Fig. 4f), suggesting that the interaction of T cells with B cells in vivo is involved in the production of autoantibodies.
In order to elucidate further the pathological role for T cells, we generated CD4-deficient SOCS-1+/ mice and analyzed their production of anti-DNA antibodies. In contrast to SOCS-1+/ mice, no elevations of anti-ssDNA antibodies were found in adult SOCS-1+/ mice lacking CD4+ T cells (Fig. 4a). This finding suggests that CD4+ T cells play a crucial role in the production of anti-DNA antibodies in SOCS-1+/ mice.
SOCS-1 is involved in the maintenance of functions of CD25+CD4+ regulatory T cells
Accumulating evidence has shown that subpopulations of CD4+ T cells, known as CD25+CD4+ regulatory T cells, potently inhibit T cell activation in vitro and play a crucial role in the maintenance of self-tolerance in vivo (2527). Recent studies suggest a possible link between SOCS-1 and these CD25+CD4+ regulatory T cells, because differential expression analyses have revealed that SOCS-1 is highly expressed in CD25+CD4+ regulatory T cells as compared with CD25CD4+ T cells (28,29). Indeed, while untreated CD25CD4+ T cells expressed only low amounts of SOCS-1 mRNA, CD25+CD4+ regulatory T cells constitutively expressed higher amounts of SOCS-1 (Fig. 5a). However, interestingly, SOCS-1 expression in regulatory T cells did not exceed the level of SOCS-1 in CD25CD4+ T cells fully stimulated with IL-4 or IFN-
(Fig. 5a). This finding suggests that SOCS-1 may be involved in the development of CD25+CD4+ regulatory T cells or may be required for the their regulatory functions upon self-reactive T cells and B cells (30,31). To investigate further the role of SOCS-1 in the development/function of regulatory T cells, we focused on the analysis of CD25+CD4+ T cells from SOCS-1+/ mice, because CD25+CD4+ T cells in Eµ-SOCS-1/ mice are likely to consist not only of regulatory T cells, but also of endogenously activated T cells (Fig. 2c).

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Fig. 5. The function of CD25+CD4+ regulatory T cells in adult SOCS-1+/ mice. (a) Constitutive expression of SOCS-1 in CD25+CD4+ regulatory T cells. Total RNA was obtained from CD25+CD4+ or CD25CD4+ T cells and SOCS-1 expression was examined by northern blot analysis. IFN- and IL-4-induced SOCS-1 expression in CD25CD4+ T cells was also shown for positive control. The band for 28S was shown for loading controls. (b) Normal regulatory T cell development in SOCS-1+/ mice. The percentage of CD25+CD4+ T cells in thymus of SOCS-1+/+ and SOCS-1+/ mice was determined by flow cytometry. (c) Purification of CD25+CD4+ T cells. Representative results of flow cytometric analysis after the purification of regulatory T cells were shown. (d) Reduced immunoregulatory capacity of SOCS-1+/ CD25+CD4+ regulatory T cells in vitro. CD25CD4+ T cells (1 x 105 cells/well) from wild-type mice were incubated with the indicated numbers of CD25+CD4+ splenocytes from wild-type or SOCS-1+/ mice in the presence of irradiated APC and anti-CD3 for 3 days. Proliferation was determined by the incorporated [3H]thymidine. (e) Impaired immunoregulatory capacity of SOCS-1+/ CD25+CD4+ regulatory T cells in the presence of IL-2. CD25CD4+ T cells (1 x 105 cells/well) from wild-type mice were cultured with CD25+CD4+ spleen cells (0.25 x 105 cells/well) from SOCS-1+/+ or SOCS-1+/ mice in the presence of irradiated APC and anti-CD3. Exogenous IL-2 was also added as indicated. Proliferation was determined by the incorporated [3H]thymidine. (f) Enhanced phosphorylation of STAT5 in response to IL-2 in SOCS-1+/ and SOCS-1/ CD25+CD4+ regulatory T cells compared to SOCS-1+/+ regulatory T cells. IL-2-induced STAT5 phosphorylation in CD25+CD4+ regulatory T cells was evaluated by western blot analysis.
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First, we examined the development of CD25+CD4+ regulatory T cells in the thymus of SOCS-1+/ mice. No differences could be detected in proportions of CD25+CD4+ regulatory T cells between adult SOCS-1+/ and control wild-type mice (Fig. 5b). Next, by use of magnetic cell sorting, we prepared CD25+CD4+ T cells (purity >90% by flow cytometry) from young, pre-onset SOCS-1+/ mice (Fig. 5c), whose CD4+ T cells expressed comparable levels of CD62L to those of SOCS-1+/+ mice (data not shown). CD25 CD4+ T cells isolated from syngeneic wild-type mice were cultured together with syngeneic wild-type APC and anti-CD3 mAb in the presence of various numbers of CD25+CD4+ regulatory T cells from young SOCS-1+/ mice, and then proliferative response was determined to explore suppressive effect of these regulatory T cells. Splenic CD25+CD4+ regulatory T cells from SOCS-1+/ mice exerted lesser suppressive effect compared to wild-type cells (Fig. 5d). Recently, it was reported that exogenous high-dose IL-2 can attenuate the suppressive action of CD25+CD4+ regulatory T cells (32,33). Reduced expression of SOCS-1 in CD25+CD4+ regulatory T cells may render these cells hyper-responsive to endogenously produced IL-2, because SOCS-1 is involved in down-regulation of IL-2 signaling (Fig. 4d) (34). Indeed, suppressive activity of SOCS-1+/ CD25+CD4+ regulatory T cells was remarkably attenuated by the addition of low doses of IL-2, compared to that of SOCS-1+/+ regulatory T cells (Fig. 5e). In addition, regulatory T cells from SOCS-1+/ mice were more sensitive to IL-2 than those from SOCS-1+/+ mice, as indicated by the clear tyrosine phosphorylation of STAT5 in SOCS-1+/ regulatory T cells after stimulation with IL-2 (Fig. 5f). As expected, IL-2-induced STAT5 phosphorylation was more easily detectable in regulatory T cells from SOCS-1/ mice (Fig. 5f). Taken together, it is conceivable that insufficient induction of SOCS-1 in CD25+CD4+ regulatory T cells allows excessive signaling of IL-2 in these populations, leads to their dysfunction and contributes to the aberrant activation of other T cells in SOCS-1+/ mice.
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Discussion
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SOCS-1 was originally cloned as a negative feedback factor of cytokine signaling. Recent analyses of SOCS-1/ mice indicate that SOCS-1 is a critical molecule for the functions of lymphocytes, especially of T cells, possibly through the regulation of cytokine signaling. In this study, we first showed that lymphocyte-specific expression of a certain level of SOCS-1 is sufficient to rescue SOCS-1/ mice from perinatal lethality, thereby indicating again the critical role of SOCS-1 in the regulation of lymphocyte functions. We next showed that SOCS-1 plays an important role in the prevention of systemic autoimmunity through the analysis of two mice models, Eµ-SOCS-1/ mice and SOCS-1+/ mice.
Although transgene-mediated expression of SOCS-1 could rescue SOCS-1/ mice from early lethality, our findings suggest that it could not fully restore the normal functions of lymphocytes. When peripheral lymphocytes in Eµ-SOCS-1/ mice were stimulated with IFN-
, SOCS-1 transgene failed to normally terminate the activation of STAT1. In addition, both T cells and B cells in Eµ-SOCS-1/ mice spontaneously exhibited an activated phenotype. Moreover, Eµ-SOCS-1/ mice exhibited multi-organ disease that partially resembled that of SOCS-1/ mice. This appears to be due to the fact that in peripheral lymphocytes, SOCS-1 expression driven by the Eµ/IgH promoter was weak (Fig. 1c) and unable to substitute the endogenous cytokine-inducible expression of SOCS-1 in normal peripheral lymphocytes.
Interestingly, however, this incomplete restoration of SOCS-1 in Eµ-SOCS-1/ mice appears to allow the development of additional pathology that has not been seen in SOCS-1/ mice. In particular, Eµ-SOCS-1/ mice spontaneously developed systemic autoimmune diseases accompanied by hypergammaglobulinemia (Fig. 3a), autoantibody production (Fig. 3b), and glomerulonephritis with mesangial cell proliferation and abnormal IgG deposition (Fig. 3c). Spontaneous development of inflammatory dermatitis (Fig. 2a and b) and pulmonary and cardiac inflammation (Fig. 2b) might strengthen the view that these mice suffer from systemic autoimmunity. The overall phenotypes of Eµ-SOCS-1/ mice resemble to those of murine lupus models (1,2).
Although the phenotypes of SOCS-1/ mice lack these lupus-like changes, this might be due to a severe reduction in the number of B cells and an early death before they could produce their own Ig (12,13). Indeed, sera from adult SOCS-1/STAT6 or SOCS-1/STAT1 double-knockout mice that can survive longer than SOCS-1/ mice contained high levels of anti-DNA antibodies (data not shown). Moreover, the screening of sera from adult SOCS-1+/ mice revealed that female SOCS-1+/ mice frequently exhibit signs of systemic autoimmunity including increased levels of autoantibodies, organ pathologies with mononuclear infiltrates and glomerulonephritis. Reduced penetrance of the disease in male SOCS-1+/ mice suggests that other factors such as female sex hormones may also be required for the pathogenesis of autoimmunity in SOCS-1+/ mice. One interesting candidate for these is prolactin, because prolactin has a capacity to augment autoimmunity (35) and its signaling is reportedly enhanced in SOCS-1+/ mice (24). In either case, it is likely that disrupted or reduced expression of SOCS-1 is a major cause for the development of systemic autoimmunity and raises the possibility that insufficient induction of SOCS-1 may participate in the development of lupus-like systemic autoimmune diseases in humans.
It has been well-established that cytokines are profoundly involved in the pathogenesis of autoimmune diseases (7), as manifested by the development of systemic autoimmunity in mice overexpressing IL-4 (3) or IFN-
(4). Thus, because SOCS-1 in vivo is a regulator of cytokines such as IFN-
(14,15), IL-4 (15), IL-12 (22,36) and prolactin (24), one can easily speculate that cytokines play important roles in the progression of systemic autoimmunity in SOCS-1 mutant mice. However, we could not simply attribute the phenotypes of these mice to the over-action of a single cytokine such as IFN-
or IL-4, because SOCS-1/STAT1 or SOCS-1/STAT6 double-knockout mice also exhibited an autoimmune phenotype (data not shown). Rather, we consider that complex actions of multiple cytokines are involved in the abnormal activation of immune systems and the progression of autoimmunity in these mutant animals.
In the absence of SOCS-1, the cytokines described above may affect a variety of cells other than lymphocytes, because SOCS-1 expression is not specific to lymphoid cells. In line with this, DC in Eµ-SOCS-1/ mice are slightly activated (Fig. 2e). Other cells such as macrophages and fibroblasts may also be affected, because these cells can express SOCS-1 in response to cytokine stimulation. Nevertheless, our findings suggest that T cells, especially CD4+ T cells, are key players for systemic autoimmunity in SOCS-1 mutant mice, since CD4+ T cells in these animals showed an activated phenotype and SOCS-1+/ mice on a CD4/ background exhibited no elevation of anti-DNA antibodies (Fig. 4a). Moreover, some nude mice transferred with pre-onset SOCS-1+/ T cells exhibit an increased production of anti-DNA antibodies (data not shown). This is also consistent with a number of previous studies demonstrating that CD4 + T cells are essential for the pathogenesis of murine lupus. It is conceivable that these excessively activated CD4+ T cells in SOCS-1 mutant mice may help B cells including those with self-reactive antibodies and cause autoimmune disease through the increased production of anti-DNA antibodies.
It is likely that several factors contribute to the excessive activation of CD4+ T cells in SOCS-1 mutant mice. For example, activated APC, including DC as mentioned earlier, appear to support the excessive activation of T cells. However, it is likely that the aberrant responses of SOCS-1 mutant T cells themselves to endogenous cytokines are also important, since SOCS-1 has a potential to inhibit T cell growth factors such as IL-2 (11,18,34). Indeed, thymocytes from pre-onset SOCS-1+/ mice exhibited increased proliferation in response to IL-2 compared to those from wild-type mice (Fig. 4d). Similarly, other factors, such as other
c-using cytokines may also contribute this process, which is supported by recent findings that SOCS-1 regulates
c cytokines including IL-2 in vivo (37). Thus, endogenous expression of cytokines such as IL-2 may support the spontaneous activation and/or proliferation of T cells in SOCS-1 mutant mice, and may induce the production of cytokines, including IL-4 and IFN-
, by these activated T cells. Because SOCS-1 mutant mice are also hyper-responsive to cytokines such as IL-4 and IFN-
(14,15), these cytokines produced by T cells can enhance pathological immune responses and may promote autoimmunity.
Our present findings also suggest that IL-2 has another mode of action in the progression of systemic autoimmunity in SOCS-1 mutant animals, which is exerted through the functional regulation of regulatory T cells. Previous studies have shown that regulatory T cells actively suppress the expansion of self-reactive T cells, thereby inhibiting the development of autoimmune diseases (2527). The majority of regulatory T cells express IL-2 receptor
chain (CD25) and, while they do not proliferate to low concentrations of IL-2 even in the presence of TCR stimulation, they do respond to high concentrations of IL-2 and lose their immunoregulatory functions (32,33). Interestingly, recent analyses have shown that increased amounts of SOCS-1 are constitutively expressed in regulatory T cells of normal mice (28,29), implying a possible role for SOCS-1 in keeping the anergic state of regulatory T cells. In line with these findings, we confirmed that CD25+CD4+ regulatory T cells preferentially express SOCS-1 without exogenous stimulation. In addition, partial loss of SOCS-1 appears to affect the function of CD25+CD4+ regulatory T cells, but not their development, because regulatory T cells from SOCS-1+/ mice in vitro exerted a lesser suppressive effect on responder T cells than did wild-type cells (Fig. 5d), despite no difference between the proportion in mutant and wild-type mice (Fig. 5c). We also showed that IL-2 can cause enhanced activation of STAT5, a major transcription factor for IL-2 signaling, in SOCS-1 mutant CD25+CD4+ regulatory T cells and can attenuate the down-regulatory action of SOCS-1+/ regulatory T cells more efficiently than that of wild-type cells (Fig. 5e). Taken together, these results suggest that one of the etiologies for systemic autoimmunity in SOCS-1 mutant mice might be the dysfunction of regulatory T cells, which might be due to enhanced IL-2 signaling in these animals. This notion may be supported by the fact that mice lacking CTLA-4, a key molecule for the function of CD25+CD4+ regulatory T cells (38), also exhibit phenotypes similar to SOCS-1/ mice (11).
It should be noted that excessive expression of SOCS-1 in regulatory T cells that completely abrogates IL-2 signaling might also be harmful, because these cells require IL-2 signaling also for their normal functional development (39,40). It is interesting to note that SOCS-1 expression in CD25+CD4+ regulatory T cells was intermediate between those of untreated CD25CD4+ T cells and fully stimulated CD25CD4+ T cells (Fig. 5a). Therefore, sufficient, but not excessive, levels of SOCS-1 may be essential for the maintenance of normal function of CD25+CD4+ regulatory T cells and normal immune response.
As indicated by the epidemiological studies, the concordance of SLE is greatly influenced by genetic factors. To date, a number of studies have proposed multiple genes that may be implicated in the pathogenesis of human SLE. Our present study of SOCS-1 mutant mice suggests that SOCS-1 can be one of those genes that influence lupus susceptibility. In this context, it should be noted that one lupus susceptibility locus localizes to proximal chromosome 16 and influences the titer of anti-dsDNA antibody (41). As the SOCS-1 gene is mapped on the same region of chromosome 16, SOCS-1 is likely to be a candidate for this lupus susceptibility locus. In addition, diminished expression of SOCS-1 as a result of epigenetic changes may also be involved in the pathogenesis of human SLE, because recent analyses of hepatocellular carcinoma cells have shown that SOCS-1 expression is silenced as a result of the methylation of CpG islands in the SOCS-1 gene (42,43). These possibilities are currently under investigation.
In conclusion, our present study re-emphasizes a critical role of SOCS-1 in the regulation of T cell activation and homeostasis, and suggests a new role of SOCS-1 in the suppression of systemic autoimmunity. Further studies are important to clarify the relevance of SOCS-1 to the development of human systemic autoimmune diseases including SLE.
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Acknowledgements
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We gratefully acknowledge the provision of STAT1/ mice by Dr R. D. Schreiber (Washington University), and STAT6/ mice by Dr S. Akira (Osaka University). We thank Dr S. Sakaguchi (Kyoto University) for helpful discussions, Mr M. Tanei, Ms M. Ishimaru (KAC Corp., Shiga, Japan) and Ms Y. Matsukawa for their expert management of the mice, and Ms M. Shimbo for secretarial assistance. This work was supported in part by a Grant-in-Aid and a Hitec Research Center Grant from the Ministry of Education, Culture, Science and Technology, Japan. H. T., T. Y. and K. N. were supported by Core Research for Evolutional Science and Technology, Japan Science and Technology Corp.
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Abbreviations
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APCantigen-presenting cell
DCdendritic cell
SLEsystemic lupus erythematosus
GCgerminal center
JAKJanus kinase
SOCSsuppressor of cytokine signaling
STATsignal transducers and activators of transcription
Tgtransgenic
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