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Complete Suppression of Insulitis and Diabetes in NOD mice Lacking Interferon Regulatory Factor-1
doi:10.1006/jaut.2001.0531, available online at http://www.idealibrary.com on
Journal of Autoimmunity (2001) 17, 119–125
Complete Suppression of Insulitis and Diabetes in NOD mice Lacking Interferon Regulatory Factor-1 Tetsuya Nakazawa1, Jo Satoh1, Kazuma Takahashi1, Yoshiyuki Sakata1, Fumiko Ikehata1, Yumiko Takizawa1, Shin-ichiro Bando1, Toshimune Housai1, Yan Li1, Chen Chen1, Takayuki Masuda2, Shigeo Kure3, Ichiro Kato4, Shin Takasawa4, Tadatsugu Taniguchi5, Hiroshi Okamoto4 and Takayoshi Toyota1 1
Division of Molecular Metabolism and Diabetes, Department of Internal Medicine, Tohoku University School of Medicine 2 Department of Medical Technology, College of Medical Science, Tohoku University 3 Department of Medical Genetics, Tohoku University School of Medicine 4 Department of Biochemistry, Tohoku University School of Medicine 5 Department of Immunology, Faculty of Medicine, University of Tokyo Received 31 May 2001 Accepted 20 October 2001 Key words: IRF-1, NOD mice, type 1 diabetes mellitus, Th1 and Th2 cytokine
Interferon regulatory factor-1 (IRF-1), a transcriptional factor, regulates type I interferon and interferon-induced genes. It was reported that IRF-1 regulates important molecules required for inflammation and immune reactions. To investigate the role of IRF-1 in the development of autoimmune diabetes, we established IRF-1 deficient (IRF-1 −/− ) non-obese diabetic (NOD) mice. IRF-1deficient C57BL/6J mice were out-crossed to NOD mice, and F1 were backcrossed to NOD mice. At the N8 generation, the heterozygote for IRF-1 mutation was intercrossed and N8F1 was obtained. Out of three NOD genotypes, IRF-1 +/+ and IRF-1 +/− developed spontaneous diabetes with an incidence of 47% (9/19) and 50% (10/20) by 30 weeks of age, respectively; whereas IRF-1 −/− did not develop diabetes (0/18, P<0.01 vs. +/+ and +/− ). Histologically, IRF-1 +/+ and IRF-1 +/− had various degrees of insulitis, but IRF-1 −/− had no insulitis. In comparison with IRF-1 +/+ , the percentage of CD4 + and Mac-1 + splenic cells significantly increased, whereas CD3 + , CD8 + and B220 + cells decreased in IRF-1 −/− . Furthermore, spleen cell proliferation in response to Con A or murine GAD65 peptide, a major autoantigen of the pancreatic -cell, significantly increased, and the IFN-/IL-10 ratio in the culture supernatant significantly decreased in IRF-1 −/− , suggesting Th2 deviation in cytokine balance. These results indicate that IRF-1 plays a key role in developing insulitis and diabetes in NOD mice. © 2001 Academic Press
Introduction
interleukin-1 converting enzyme (ICE) [7]. In addition, IRF-1 plays important roles in differentiation and function of immune cells such as CD8 cells [8, 9], development of the Th1 response [9, 10, 11], and natural killer (NK) cell differentiation [12]. Furthermore, a role of IRF-1 has been indicated in autoimmune disease, i.e., incidence and severity were reduced in collagen-induced arthritis and experimental allergic encephalomyelitis in IRF-1-deficient mice [13]. Previously, it was reported that the expression of interferon- (IFN-) in the pancreatic -cells induces immune-mediated insulin-dependent diabetes mellitus (IDDM) and neutralization of IFN- with antibody prevents development of IDDM in transgenic mice [14]. Considering these reports on roles of IRF-1 in the immune system along with the pathogenesis of autoimmune diabetes, we assumed that IRF-1 plays a dominant role in anti-islet autoimmunity. In this study we created IRF-1 deficient NOD mice to evaluate the role of IRF-1 in the development of insulitis and diabetes.
In NOD mice, a model of type 1 diabetes mellitus, the insulin-producing pancreatic -cell is selectively destroyed by T cell-mediated autoimmunity. Various immune interventions of the development and action of effector cells for -cell destruction prevent the development of diabetes in NOD mice [1, 2]. The interferon regulatory factor-1 (IRF-1) family, which was originally described as a positive and negative regulator of type I interferon and interferoninducible genes, is a member of a multigene family of transcription factors [3]. Signaling via interferon receptors promotes transcription of IRF-1, and this molecule controls the expression of interferonresponsive genes, including HLA class I and class II [4, 5], inducible nitric oxide synthase (iNOS) [6], and Correspondence to: Dr Jo Satoh, Division of Molecular Metabolism and Diabetes, Department of Internal Medicine, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan. Fax +81-22-717-7177. E-mail: [email protected] 119 0896–8411/01/060119+07 $35.00/0
© 2001 Academic Press
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Materials and Methods Mice IRF-1-deficient (IRF-1 −/− ) mice (C57BL/6J background) [9] were kindly provided by Professor Tadatsugu Taniguchi, Tokyo University. NOD/Shi mice were obtained from Clea Japan (Tokyo, Japan) and were maintained by continued intercross under specific pathogen-free conditions in the Institute for Experimental Animals, Tohoku University School of Medicine. Principles of laboratory animal care (NIH publication no. 85-23, revised 1985) were adhered to this study.
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Histological examination Mice were killed by cervical dislocation under ether anesthesia, and pancreas was obtained at 15 weeks of age and fixed in neutral-buffered formalin. Nonconsecutive paraffin-embedded sections were stained with hematoxylin and eosin. The number of islets was counted and sections were scored for insulitis by two investigators in a blind fashion. Insulitis scores were graded as 0, no mononuclear cell infiltration; 1, periinsulitis; 2, mononuclear cell infiltration in <50% of the islet area; or 3, mononuclear cell infiltration in ≥50% of the islet area. Flow cytometric analysis
Generation of IRF-1 −/− NOD mice and genotyping of IRF-1 allele IRF-1-deficient (heterozygote) mice were outcrossed to NOD mice (Kd, I-Ag7, I-Enull, Db) and a F1 male was backcrossed into NOD. In each generation, mice were typed by polymerase chain reaction (PCR) with two sets of primers within wild-type IRF-1 gene (sense primer, IRF-1-I, 5′-TTCCAGATTCCATGGAAGCA CGC-3′ on exon 3 and reverse primer, IRF-1-II, 5′-ATGGCACAACGGAAGTTTGCC-3′ on exon 4) and neomycin resistance gene (neo′) of the targeted locus (sense primer, IRF-1-I, 5′-TTCCAGATTCC ATGGAAGCACGC-3′ on exon 3 and reverse primer, IRF-1-III, 5′ATTCGCCAATGACAAGACGCTGG-3′ on neo′) [9]. Each yielded a band of approximate 0.9 kbp of the wild-type and 0.7 kbp of the targeted locus, respectively. IRF-1 +/+ , +/− and −/− NOD mice were generated by intercrossing eighth backcross (N9) generation of IRF-1 +/− NOD mice.
Analysis of microsatellite markers To examine the substitution of IDDM susceptibility (Idd) genes, which are reported to link to insulitis and diabetes [15], in IRF-1 deficient NOD mice, genomes of N7 were assessed with locus-specific microsatellite markers for Idd1 (D17Mit34), Idd3 (D3Mit95), Idd5 (D1Mit5), Idd10 (D3Mit103), and Idd4 (D11Mit320) which is located on the same chromosome (Chromosome 11) as for IRF-1 [9]. Genomes of NOD mice were also assessed similarly.
The spleen cells from IRF-1 +/+ and IRF-1 −/− NOD mice were stained with a panel of mouse monoclonal antibodies; FITC-labeled anti-CD3 (145-2C11, PharMingen, San Diego, CA, USA), PE-labeled antiCD4 (RM4-5, PharMingen), FITC-labeled anti-CD8 (53-6.7, PharMingen), FITC-labeled anti-CD11b (Mac 1) (M1/70, CEDARLANE, Ontario, Canada), or PE-labeled anti-CD45R (B220) (RA3-6B2, PharMingen), and analyzed by a FACSCalibury (Becton Dickinson and Co, Mountain View, CA, USA). Ten thousand cells were counted. Data acquisition and analysis were performed with the CellQuest Software (Becton Dickinson and Co). Proliferation and cytokine assay The same spleen cells used for the flowcytometry were resuspended in RPMI-1640 (GIBCO BRL, Grand Island, NY, USA) and supplemented with 10% fetal calf serum (ICN Biomedicals, Inc., Aurora, OH, USA), sodium pyruvate, and a mixture of streptomycin, penicillin and fungison; 2×106/ml of the cells were then cultured with concanavalin A (Con A) or peptide of murine glutamic acid decarboxylase 65 (see below) in 96-well round-bottom microtiter plates (200 ml/ well). Proliferation was measured on day 3 for Con A and day 5 for GAD65 peptide by a 6 h pulse label with [3H] thymidine (0.5 Ci/well, Amersham Life Science, Buckingham, UK). IFN- and IL-10 concentrations of the supernatant were measured using Cytoscreeny immunoassay kits (Biosource International, Camarillo, CA, USA).
Detection of diabetes
Murine glutamic acid decarboxylase 65 (GAD65) peptide
Diabetes was monitored once or twice a week by detecting the presence of urine glucose using Testape A (Eli Lilly & Co., Indianapolis, IN, USA) as previously reported [16]. Mice were defined as diabetic when they showed persistent glucosuria. Only female mice were used for experiments on diabetes incidence and histological and immunological analysis because of their high incidence of diabetes.
A murine cDNA fragment encoding a murine GAD65 C-terminal peptide (86 amino acids) was obtained using reverse transcription-mediated PCR, and expressed in Escherischia coli using T7 polymerase and T7 promoter (pET system, Novagen, Madison, WI, USA) [17]. Endotoxin contamination in the purified peptide solution was negligible in the proliferation assay.
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Table 1. Analysis of microsatellite markers linked to Idd loci Idd locus/chromosome Microsatellite marker N7 male
Idd1/17
Idd3/3
Idd4/11
Idd5/1
Idd10/3
D17Mit34
D3Mit95
D11Mit320
D1Mit5
D3Mit103
D/D
D/D
D/D
D/D
D/D
D/D, NOD homozygote at the indicated locus.
Statistical analysis
60
Results Analysis of microsatellite markers Male and female heterozygous carriers of the IRF-1deficient allele from the N7 backcross generation were assessed by PCR for microsatellite markers. As shown in Table 1, markers linked to Idd1, Idd3, Idd4, Idd5 and Idd10 loci were all homozygous for NOD alleles, indicating that those Idd loci were preserved during the backcrossing. These mice were served as the progenitor for all mice in subsequent backcross generations.
IRF-1 deficiency completely suppressed insulitis and diabetes in NOD mice Diabetes developed from 13–16 weeks of age, and by 30 weeks of age, diabetes incidence reached approximately 50% in IRF-1 +/+ and +/− NOD mice, whereas none of IRF-1 −/− mice developed diabetes (Figure 1). There was no difference in body weight among IRF1 +/+ , +/− and −/− NOD mice and no abnormal appearances in IRF +/− and −/− mice before the onset of diabetes. Only female mice were used for this and subsequent experiments because of their high incidence of diabetes. Severity of insulitis was evaluated with the insulitis score at 15 weeks of age. Various degrees of insulitis existed in IRF-1 +/+ and +/− mice, whereas no insulitis was observed in IRF-1 −/− mice (Table 2).
Flow cytometric analysis of surface molecule expression on spleen cells from IRF-1 +/+ and NOD mice
−/−
In order to determine the immune mechanism in the suppression of autoimmune diabetes, spleen cell populations were analysed in IRF-1 +/+ and −/− NOD mice by a flow cytometry, because it was reported that the IRF-1 −/− mice have massive defects in the generation of T cells [9]. The spleen cells from IRF-1 −/−
50 Incidence of diabetes (%)
Statistical significance between sets of data was assessed using the -square test or the Mann-Whitney test.
40
30
20
10
0
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 Weeks of age
Figure 1. Cumulative incidence of diabetes in IRF-1 +/+ , +/− , and −/− NOD mice. Female mice were examined for glucosuria once a week. Open circle; IRF-1 +/+ NOD (n=19); open square, IRF-1 +/− NOD (n=20); Closed square, IRF-1 −/− NOD (n=19). P<0.01: IRF-1 −/− NOD vs. IRF-1 +/+ and IRF-1 +/− NOD.
mice had significantly more CD4 + and MAC-1 + cells, and significantly less CD3 + , CD8 + , and B220 + cells than those from IRF-1 +/+ mice (Figure 2). Total spleen cell number did not significantly differ between IRF1 +/+ and −/− NOD mice (data not shown).
Proliferation and cytokine production in response to ConA or GAD65 peptide of spleen cells from IRF-1 +/+ and −/− NOD mice Next, to observe the function of the spleen cells in IRF-1 +/+ and −/− NOD mice, we examined proliferation and cytokine production by spleen cells in response to Con A and GAD65 peptide, a major autoantigen of the pancreatic -cell in type 1 diabetes [1, 2]. The cells from IRF-1 −/− mice displayed a significantly higher proliferative response to Con A or GAD65-derived peptide than those from IRF-1 +/+ mice (Figure 3). The response to 5 g/ml of Con A was far lower than the response to 1 g/ml in this experiment, although a reason is unclear. Supernatant from the same series of the culture as for the proliferation assay was measured for IFN- and IL-10. Spleen cells from IRF-1 −/− mice produced
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Table 2. Reduced insulitis score in islets from IRF-1 −/− NOD mice. Islets from 15-week-old IRF-1 +/+ , +/− , and −/− mice were assigned to histological scores as described in Methods. The statistical analysis was performed by 2-test NOD mice
Surface molecules
IRF-1 +/+ IRF-1 +/− IRF-1 −/−
Insulitis score
No. of islets counted
0
1
2
3
Mean
P value (vs +/+ )
54 79 36
31 36 36
10 14 0
5 15 0
8 14 0
0.8 1.1 0
— 0.14 <0.0001
CD3
*
CD4
*
CD8
*
B220
*
Mac 1
*
0
10
20
30
40
50
% positive cells
Figure 2. Flow cytometric analysis of surface molecule expression on spleen cells from IRF-1 +/+ and −/− NOD mice. Pooled spleen cells were obtained from three IRF-1 +/+ and −/− NOD mice at 30 weeks of age. The data are the mean±1 SD of triplicate samples. Closed column, IRF-1 +/+ NOD mice; hatched column, IRF-1 −/− NOD mice; *, P<0.05 (Mann-Whitney test).
significantly less IFN- and more IL-10 in the stimulation with Con A (Figure 4A), whereas the cells produced significantly less IFN- and IL-10 with GAD65-derived peptide, as compared with those from IRF-1 +/+ mice (Figure 4B). However, the IFN-/ IL-10 ratio was significantly reduced in both Con A and GAD65 cases (Figure 4A and 4B).
Discussion In IRF-1 −/− NOD mice, development of insulitis and diabetes were completely suppressed as compared to those in IRF-1 +/− and IRF-1 +/+ NOD mice. The lack of insulitis is not due to the absence of the Idd genes, because all of the microsatellite markers that are
reported to link to insulitis and diabetes [15] were NOD mouse types. This suggests that deficiency of IRF-1 gene per se is responsible for the lack of insulitis and diabetes in IRF-1 −/− NOD mice. The lack of insulitis indicates that induction of autoimmunity and/or differentiation of effector cells against the pancreatic -cell might be suppressed, as reported in Fas-deficient NOD mice [18] and anti-CD40 mAbtreated NOD mice [19]. Although the precise mechanism(s) of the suppression in the induction of autoimmunity and/or differentiation of the effector cells was not clarified in our study, some possibilities are implicated in the literature and our results. Type 1 diabetes in NOD mice is a T cell-mediated autoimmune disease [2]. Briefly, CD4 + T cells reactive with -cell antigens such as GAD are generated, and CD4 + and CD8 + T cells and macrophages infiltrate into the islets (insulitis). In the insulitis lesion, various cytokines are produced by T cells and macrophages, and CD4 + and CD8 + effector T cells are generated under Th1 cytokine (IL-12 and INF-)-dominant environment and selectively destroy -cells [2]. In addition, IL-1 and IFN- promote -cell destruction by upregulating inducible nitric oxide synthase (iNOS) expression and NO production by macrophages and -cells [20]. Most of these autoimmune mechanisms in NOD mice are indicated to be defective in IRF-1-deficient mice as discussed below. MHC class I expression, necessary for T cell development, is inefficient in the thymic environment in IRF-1 deficient mice [9, 21], and immature T cells are able to develop into CD4 + but not efficiently into CD8 + T cells [8], which are the major effector cells for the -cell destruction in NOD mice [1, 2]. Furthermore, the absence of IRF-1 leads to the defect in the development of the Th1 type immune response and to the inappropriate induction of the Th2 type immune response, which are associated with multiple defects in CD4 + T cells [22] and NK cells [12], and in induction of IL-12 by macrophages [23]. These defects in IRF-1-deficient mice are comparible to those shown in our IRF-1-deficient NOD mice, e.g., relatively increased number of CD4 + cells, increased proliferative response of spleen cells to Con A and GAD65 peptide, a major antigen of the -cell, probably due to an increased number of CD4 + cells, decreased number of CD8 + cells and changes in cytokine production. CD8 + cells are necessary for both the initiation and
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A
B
Con A 1 µg/ml
*
GAD peptide 10 µg/ml
*
Con A 5 µg/ml
*
GAD peptide 50 µg/ml
*
0
20
40
60
80
0
3
2
4
6
8
3
H -thymidine uptake (× 10 cpm)
Figure 3. Proliferative response by spleen cells from IRF-1 +/+ and −/− NOD mice to Con A (panel A) or a GAD65-derived peptide (panel B). Pooled spleen cells were obtained from three IRF-1 +/+ and −/− NOD mice at 30 weeks of age. The cells were cultured in the presence of Con A (panel A) or a GAD-derived peptide (panel B). The data are the mean±1 SD of triplicate wells. Closed column, IRF-1 +/+ NOD mice; hatched column, IRF-1 −/− NOD mice; *, P<0.05 (Mann-Whitney test).
A Spleen cells Con A
+ +
/
1 µg/ml
– –
5 µg/ml
/
400
IFN-γ (pg/ml)
300
200
100
IL-10 (pg/ml)
0 0
100
IFN-γ /IL-10
200
300 0
10
100
150
B Spleen GAD cells peptide
+ +
/
10 µg/ml
– –
50 µg/ml
/
400
IFN-γ (pg/ml)
300
200
100
IL-10 (pg/ml)
0 0
20
40 +/+
60
IFN-γ /IL-10
80
100 0
1
2
3
4
5
6
−/−
Figure 4. IFN- and IL-10 production by spleen cells from IRF-1 and NOD mice in the presence of Con A (panel A) or a GAD-derived peptide (panel B). Pooled spleen cells were obtained from three IRF-1 +/+ and −/− NOD mice at 30 weeks of age. The cells were cultured in the presence of Con A (panel A) or a GAD-derived peptide (panel B). An IFN-/IL-10 ratio was calculated by dividing the mean IFN- concentration by the mean IL-10 concentration in triplicate wells. Closed column, IRF-1 +/+ NOD mice; hatched column, IRF-1 −/− NOD mice; *, P<0.05 (Mann-Whitney test).
effector phases of type 1 diabetes in NOD mice [24]. IFN- production decreased and IL-10 production increased with Con A stimulation in IRF-1 −/− NOD mice, whereas both productions decreased with
GAD65-peptide stimulation. Although the reason for the difference between the cytokine responses to Con A and GAD65 peptide is unclear, IFN- production and the ratio of Th1/Th2 cytokine (IFN-/IL-10)
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productions significantly decreased in both cases, suggesting an immune deviation to the Th2 type response. The decreased production of IFN- may have protected -cells from autoimmune destruction, because various reports indicate that the development of autoimmune diabetes in NOD mice is accelerated under the predominance of Th1 cytokines [2], and because the decreased production of IFN- downregulates iNOS expression and production of NO, one of mediators of -cell destruction [20], in macrophages and -cells. Actually it has been reported that iNOS induction is impaired in IRF-1-deficient mice [6]. In addition to the decreased IFN- production, defective IFN--mediated immune responses via IRF-1 may have impaired autoimmune pathways in NOD mice. On the other hand, it has been indicated that IFN- and IFN- receptor system does not profoundly influence NOD mouse diabetes. Genetic absence of IFN- delays but does not prevent diabetes in NOD mice [25]. Lack of IFN- receptor confers resistance to cyclophosphamide-induced diabetes without affecting the natural course of diabetes development in NOD mice [26]. Although IRF-1 deficiency completely prevented diabetes in NOD mice, the influence of defect in IFN- and IFN-R system is limited [25, 26]. This discrepancy is understandable, because IRF-1, one of key molecules in host defense and immune system, is induced not only by type II IFN (IFN-) but also by type I IFNs (IFN- and IFN-) [27]. Thus, it is conceivable that multiple mechanisms mediated by IRF-1 may have prevented the development of autoimmune diabetes in IRF-1-deficient NOD mice. We have shown that the knockout of IRF-1 gene expression completely suppressed the development of autoimmune diabetes, although the exact immune mechanisms remain to be elucidated. We conclude that the signal via IRF-1 plays a pivotal role in the development of anti-islet autoimmunity in NOD mice.
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Acknowledgements
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This work was supported in part by Grants-in-Aid for Scientific Research (10671053) from the Ministry of Education, Science and Culture, Japan. The results of this study were presented in a preliminary form at the Annual Meeting of the American Diabetes Association, Chicago, June 1998.
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Journal of Autoimmunity (2001) 17, 119–125
Complete Suppression of Insulitis and Diabetes in NOD mice Lacking Interferon Regulatory Factor-1 Tetsuya Nakazawa1, Jo Satoh1, Kazuma Takahashi1, Yoshiyuki Sakata1, Fumiko Ikehata1, Yumiko Takizawa1, Shin-ichiro Bando1, Toshimune Housai1, Yan Li1, Chen Chen1, Takayuki Masuda2, Shigeo Kure3, Ichiro Kato4, Shin Takasawa4, Tadatsugu Taniguchi5, Hiroshi Okamoto4 and Takayoshi Toyota1 1
Division of Molecular Metabolism and Diabetes, Department of Internal Medicine, Tohoku University School of Medicine 2 Department of Medical Technology, College of Medical Science, Tohoku University 3 Department of Medical Genetics, Tohoku University School of Medicine 4 Department of Biochemistry, Tohoku University School of Medicine 5 Department of Immunology, Faculty of Medicine, University of Tokyo Received 31 May 2001 Accepted 20 October 2001 Key words: IRF-1, NOD mice, type 1 diabetes mellitus, Th1 and Th2 cytokine
Interferon regulatory factor-1 (IRF-1), a transcriptional factor, regulates type I interferon and interferon-induced genes. It was reported that IRF-1 regulates important molecules required for inflammation and immune reactions. To investigate the role of IRF-1 in the development of autoimmune diabetes, we established IRF-1 deficient (IRF-1 −/− ) non-obese diabetic (NOD) mice. IRF-1deficient C57BL/6J mice were out-crossed to NOD mice, and F1 were backcrossed to NOD mice. At the N8 generation, the heterozygote for IRF-1 mutation was intercrossed and N8F1 was obtained. Out of three NOD genotypes, IRF-1 +/+ and IRF-1 +/− developed spontaneous diabetes with an incidence of 47% (9/19) and 50% (10/20) by 30 weeks of age, respectively; whereas IRF-1 −/− did not develop diabetes (0/18, P<0.01 vs. +/+ and +/− ). Histologically, IRF-1 +/+ and IRF-1 +/− had various degrees of insulitis, but IRF-1 −/− had no insulitis. In comparison with IRF-1 +/+ , the percentage of CD4 + and Mac-1 + splenic cells significantly increased, whereas CD3 + , CD8 + and B220 + cells decreased in IRF-1 −/− . Furthermore, spleen cell proliferation in response to Con A or murine GAD65 peptide, a major autoantigen of the pancreatic -cell, significantly increased, and the IFN-/IL-10 ratio in the culture supernatant significantly decreased in IRF-1 −/− , suggesting Th2 deviation in cytokine balance. These results indicate that IRF-1 plays a key role in developing insulitis and diabetes in NOD mice. © 2001 Academic Press
Introduction
interleukin-1 converting enzyme (ICE) [7]. In addition, IRF-1 plays important roles in differentiation and function of immune cells such as CD8 cells [8, 9], development of the Th1 response [9, 10, 11], and natural killer (NK) cell differentiation [12]. Furthermore, a role of IRF-1 has been indicated in autoimmune disease, i.e., incidence and severity were reduced in collagen-induced arthritis and experimental allergic encephalomyelitis in IRF-1-deficient mice [13]. Previously, it was reported that the expression of interferon- (IFN-) in the pancreatic -cells induces immune-mediated insulin-dependent diabetes mellitus (IDDM) and neutralization of IFN- with antibody prevents development of IDDM in transgenic mice [14]. Considering these reports on roles of IRF-1 in the immune system along with the pathogenesis of autoimmune diabetes, we assumed that IRF-1 plays a dominant role in anti-islet autoimmunity. In this study we created IRF-1 deficient NOD mice to evaluate the role of IRF-1 in the development of insulitis and diabetes.
In NOD mice, a model of type 1 diabetes mellitus, the insulin-producing pancreatic -cell is selectively destroyed by T cell-mediated autoimmunity. Various immune interventions of the development and action of effector cells for -cell destruction prevent the development of diabetes in NOD mice [1, 2]. The interferon regulatory factor-1 (IRF-1) family, which was originally described as a positive and negative regulator of type I interferon and interferoninducible genes, is a member of a multigene family of transcription factors [3]. Signaling via interferon receptors promotes transcription of IRF-1, and this molecule controls the expression of interferonresponsive genes, including HLA class I and class II [4, 5], inducible nitric oxide synthase (iNOS) [6], and Correspondence to: Dr Jo Satoh, Division of Molecular Metabolism and Diabetes, Department of Internal Medicine, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan. Fax +81-22-717-7177. E-mail: [email protected] 119 0896–8411/01/060119+07 $35.00/0
© 2001 Academic Press
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Materials and Methods Mice IRF-1-deficient (IRF-1 −/− ) mice (C57BL/6J background) [9] were kindly provided by Professor Tadatsugu Taniguchi, Tokyo University. NOD/Shi mice were obtained from Clea Japan (Tokyo, Japan) and were maintained by continued intercross under specific pathogen-free conditions in the Institute for Experimental Animals, Tohoku University School of Medicine. Principles of laboratory animal care (NIH publication no. 85-23, revised 1985) were adhered to this study.
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Histological examination Mice were killed by cervical dislocation under ether anesthesia, and pancreas was obtained at 15 weeks of age and fixed in neutral-buffered formalin. Nonconsecutive paraffin-embedded sections were stained with hematoxylin and eosin. The number of islets was counted and sections were scored for insulitis by two investigators in a blind fashion. Insulitis scores were graded as 0, no mononuclear cell infiltration; 1, periinsulitis; 2, mononuclear cell infiltration in <50% of the islet area; or 3, mononuclear cell infiltration in ≥50% of the islet area. Flow cytometric analysis
Generation of IRF-1 −/− NOD mice and genotyping of IRF-1 allele IRF-1-deficient (heterozygote) mice were outcrossed to NOD mice (Kd, I-Ag7, I-Enull, Db) and a F1 male was backcrossed into NOD. In each generation, mice were typed by polymerase chain reaction (PCR) with two sets of primers within wild-type IRF-1 gene (sense primer, IRF-1-I, 5′-TTCCAGATTCCATGGAAGCA CGC-3′ on exon 3 and reverse primer, IRF-1-II, 5′-ATGGCACAACGGAAGTTTGCC-3′ on exon 4) and neomycin resistance gene (neo′) of the targeted locus (sense primer, IRF-1-I, 5′-TTCCAGATTCC ATGGAAGCACGC-3′ on exon 3 and reverse primer, IRF-1-III, 5′ATTCGCCAATGACAAGACGCTGG-3′ on neo′) [9]. Each yielded a band of approximate 0.9 kbp of the wild-type and 0.7 kbp of the targeted locus, respectively. IRF-1 +/+ , +/− and −/− NOD mice were generated by intercrossing eighth backcross (N9) generation of IRF-1 +/− NOD mice.
Analysis of microsatellite markers To examine the substitution of IDDM susceptibility (Idd) genes, which are reported to link to insulitis and diabetes [15], in IRF-1 deficient NOD mice, genomes of N7 were assessed with locus-specific microsatellite markers for Idd1 (D17Mit34), Idd3 (D3Mit95), Idd5 (D1Mit5), Idd10 (D3Mit103), and Idd4 (D11Mit320) which is located on the same chromosome (Chromosome 11) as for IRF-1 [9]. Genomes of NOD mice were also assessed similarly.
The spleen cells from IRF-1 +/+ and IRF-1 −/− NOD mice were stained with a panel of mouse monoclonal antibodies; FITC-labeled anti-CD3 (145-2C11, PharMingen, San Diego, CA, USA), PE-labeled antiCD4 (RM4-5, PharMingen), FITC-labeled anti-CD8 (53-6.7, PharMingen), FITC-labeled anti-CD11b (Mac 1) (M1/70, CEDARLANE, Ontario, Canada), or PE-labeled anti-CD45R (B220) (RA3-6B2, PharMingen), and analyzed by a FACSCalibury (Becton Dickinson and Co, Mountain View, CA, USA). Ten thousand cells were counted. Data acquisition and analysis were performed with the CellQuest Software (Becton Dickinson and Co). Proliferation and cytokine assay The same spleen cells used for the flowcytometry were resuspended in RPMI-1640 (GIBCO BRL, Grand Island, NY, USA) and supplemented with 10% fetal calf serum (ICN Biomedicals, Inc., Aurora, OH, USA), sodium pyruvate, and a mixture of streptomycin, penicillin and fungison; 2×106/ml of the cells were then cultured with concanavalin A (Con A) or peptide of murine glutamic acid decarboxylase 65 (see below) in 96-well round-bottom microtiter plates (200 ml/ well). Proliferation was measured on day 3 for Con A and day 5 for GAD65 peptide by a 6 h pulse label with [3H] thymidine (0.5 Ci/well, Amersham Life Science, Buckingham, UK). IFN- and IL-10 concentrations of the supernatant were measured using Cytoscreeny immunoassay kits (Biosource International, Camarillo, CA, USA).
Detection of diabetes
Murine glutamic acid decarboxylase 65 (GAD65) peptide
Diabetes was monitored once or twice a week by detecting the presence of urine glucose using Testape A (Eli Lilly & Co., Indianapolis, IN, USA) as previously reported [16]. Mice were defined as diabetic when they showed persistent glucosuria. Only female mice were used for experiments on diabetes incidence and histological and immunological analysis because of their high incidence of diabetes.
A murine cDNA fragment encoding a murine GAD65 C-terminal peptide (86 amino acids) was obtained using reverse transcription-mediated PCR, and expressed in Escherischia coli using T7 polymerase and T7 promoter (pET system, Novagen, Madison, WI, USA) [17]. Endotoxin contamination in the purified peptide solution was negligible in the proliferation assay.
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Table 1. Analysis of microsatellite markers linked to Idd loci Idd locus/chromosome Microsatellite marker N7 male
Idd1/17
Idd3/3
Idd4/11
Idd5/1
Idd10/3
D17Mit34
D3Mit95
D11Mit320
D1Mit5
D3Mit103
D/D
D/D
D/D
D/D
D/D
D/D, NOD homozygote at the indicated locus.
Statistical analysis
60
Results Analysis of microsatellite markers Male and female heterozygous carriers of the IRF-1deficient allele from the N7 backcross generation were assessed by PCR for microsatellite markers. As shown in Table 1, markers linked to Idd1, Idd3, Idd4, Idd5 and Idd10 loci were all homozygous for NOD alleles, indicating that those Idd loci were preserved during the backcrossing. These mice were served as the progenitor for all mice in subsequent backcross generations.
IRF-1 deficiency completely suppressed insulitis and diabetes in NOD mice Diabetes developed from 13–16 weeks of age, and by 30 weeks of age, diabetes incidence reached approximately 50% in IRF-1 +/+ and +/− NOD mice, whereas none of IRF-1 −/− mice developed diabetes (Figure 1). There was no difference in body weight among IRF1 +/+ , +/− and −/− NOD mice and no abnormal appearances in IRF +/− and −/− mice before the onset of diabetes. Only female mice were used for this and subsequent experiments because of their high incidence of diabetes. Severity of insulitis was evaluated with the insulitis score at 15 weeks of age. Various degrees of insulitis existed in IRF-1 +/+ and +/− mice, whereas no insulitis was observed in IRF-1 −/− mice (Table 2).
Flow cytometric analysis of surface molecule expression on spleen cells from IRF-1 +/+ and NOD mice
−/−
In order to determine the immune mechanism in the suppression of autoimmune diabetes, spleen cell populations were analysed in IRF-1 +/+ and −/− NOD mice by a flow cytometry, because it was reported that the IRF-1 −/− mice have massive defects in the generation of T cells [9]. The spleen cells from IRF-1 −/−
50 Incidence of diabetes (%)
Statistical significance between sets of data was assessed using the -square test or the Mann-Whitney test.
40
30
20
10
0
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 Weeks of age
Figure 1. Cumulative incidence of diabetes in IRF-1 +/+ , +/− , and −/− NOD mice. Female mice were examined for glucosuria once a week. Open circle; IRF-1 +/+ NOD (n=19); open square, IRF-1 +/− NOD (n=20); Closed square, IRF-1 −/− NOD (n=19). P<0.01: IRF-1 −/− NOD vs. IRF-1 +/+ and IRF-1 +/− NOD.
mice had significantly more CD4 + and MAC-1 + cells, and significantly less CD3 + , CD8 + , and B220 + cells than those from IRF-1 +/+ mice (Figure 2). Total spleen cell number did not significantly differ between IRF1 +/+ and −/− NOD mice (data not shown).
Proliferation and cytokine production in response to ConA or GAD65 peptide of spleen cells from IRF-1 +/+ and −/− NOD mice Next, to observe the function of the spleen cells in IRF-1 +/+ and −/− NOD mice, we examined proliferation and cytokine production by spleen cells in response to Con A and GAD65 peptide, a major autoantigen of the pancreatic -cell in type 1 diabetes [1, 2]. The cells from IRF-1 −/− mice displayed a significantly higher proliferative response to Con A or GAD65-derived peptide than those from IRF-1 +/+ mice (Figure 3). The response to 5 g/ml of Con A was far lower than the response to 1 g/ml in this experiment, although a reason is unclear. Supernatant from the same series of the culture as for the proliferation assay was measured for IFN- and IL-10. Spleen cells from IRF-1 −/− mice produced
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Table 2. Reduced insulitis score in islets from IRF-1 −/− NOD mice. Islets from 15-week-old IRF-1 +/+ , +/− , and −/− mice were assigned to histological scores as described in Methods. The statistical analysis was performed by 2-test NOD mice
Surface molecules
IRF-1 +/+ IRF-1 +/− IRF-1 −/−
Insulitis score
No. of islets counted
0
1
2
3
Mean
P value (vs +/+ )
54 79 36
31 36 36
10 14 0
5 15 0
8 14 0
0.8 1.1 0
— 0.14 <0.0001
CD3
*
CD4
*
CD8
*
B220
*
Mac 1
*
0
10
20
30
40
50
% positive cells
Figure 2. Flow cytometric analysis of surface molecule expression on spleen cells from IRF-1 +/+ and −/− NOD mice. Pooled spleen cells were obtained from three IRF-1 +/+ and −/− NOD mice at 30 weeks of age. The data are the mean±1 SD of triplicate samples. Closed column, IRF-1 +/+ NOD mice; hatched column, IRF-1 −/− NOD mice; *, P<0.05 (Mann-Whitney test).
significantly less IFN- and more IL-10 in the stimulation with Con A (Figure 4A), whereas the cells produced significantly less IFN- and IL-10 with GAD65-derived peptide, as compared with those from IRF-1 +/+ mice (Figure 4B). However, the IFN-/ IL-10 ratio was significantly reduced in both Con A and GAD65 cases (Figure 4A and 4B).
Discussion In IRF-1 −/− NOD mice, development of insulitis and diabetes were completely suppressed as compared to those in IRF-1 +/− and IRF-1 +/+ NOD mice. The lack of insulitis is not due to the absence of the Idd genes, because all of the microsatellite markers that are
reported to link to insulitis and diabetes [15] were NOD mouse types. This suggests that deficiency of IRF-1 gene per se is responsible for the lack of insulitis and diabetes in IRF-1 −/− NOD mice. The lack of insulitis indicates that induction of autoimmunity and/or differentiation of effector cells against the pancreatic -cell might be suppressed, as reported in Fas-deficient NOD mice [18] and anti-CD40 mAbtreated NOD mice [19]. Although the precise mechanism(s) of the suppression in the induction of autoimmunity and/or differentiation of the effector cells was not clarified in our study, some possibilities are implicated in the literature and our results. Type 1 diabetes in NOD mice is a T cell-mediated autoimmune disease [2]. Briefly, CD4 + T cells reactive with -cell antigens such as GAD are generated, and CD4 + and CD8 + T cells and macrophages infiltrate into the islets (insulitis). In the insulitis lesion, various cytokines are produced by T cells and macrophages, and CD4 + and CD8 + effector T cells are generated under Th1 cytokine (IL-12 and INF-)-dominant environment and selectively destroy -cells [2]. In addition, IL-1 and IFN- promote -cell destruction by upregulating inducible nitric oxide synthase (iNOS) expression and NO production by macrophages and -cells [20]. Most of these autoimmune mechanisms in NOD mice are indicated to be defective in IRF-1-deficient mice as discussed below. MHC class I expression, necessary for T cell development, is inefficient in the thymic environment in IRF-1 deficient mice [9, 21], and immature T cells are able to develop into CD4 + but not efficiently into CD8 + T cells [8], which are the major effector cells for the -cell destruction in NOD mice [1, 2]. Furthermore, the absence of IRF-1 leads to the defect in the development of the Th1 type immune response and to the inappropriate induction of the Th2 type immune response, which are associated with multiple defects in CD4 + T cells [22] and NK cells [12], and in induction of IL-12 by macrophages [23]. These defects in IRF-1-deficient mice are comparible to those shown in our IRF-1-deficient NOD mice, e.g., relatively increased number of CD4 + cells, increased proliferative response of spleen cells to Con A and GAD65 peptide, a major antigen of the -cell, probably due to an increased number of CD4 + cells, decreased number of CD8 + cells and changes in cytokine production. CD8 + cells are necessary for both the initiation and
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A
B
Con A 1 µg/ml
*
GAD peptide 10 µg/ml
*
Con A 5 µg/ml
*
GAD peptide 50 µg/ml
*
0
20
40
60
80
0
3
2
4
6
8
3
H -thymidine uptake (× 10 cpm)
Figure 3. Proliferative response by spleen cells from IRF-1 +/+ and −/− NOD mice to Con A (panel A) or a GAD65-derived peptide (panel B). Pooled spleen cells were obtained from three IRF-1 +/+ and −/− NOD mice at 30 weeks of age. The cells were cultured in the presence of Con A (panel A) or a GAD-derived peptide (panel B). The data are the mean±1 SD of triplicate wells. Closed column, IRF-1 +/+ NOD mice; hatched column, IRF-1 −/− NOD mice; *, P<0.05 (Mann-Whitney test).
A Spleen cells Con A
+ +
/
1 µg/ml
– –
5 µg/ml
/
400
IFN-γ (pg/ml)
300
200
100
IL-10 (pg/ml)
0 0
100
IFN-γ /IL-10
200
300 0
10
100
150
B Spleen GAD cells peptide
+ +
/
10 µg/ml
– –
50 µg/ml
/
400
IFN-γ (pg/ml)
300
200
100
IL-10 (pg/ml)
0 0
20
40 +/+
60
IFN-γ /IL-10
80
100 0
1
2
3
4
5
6
−/−
Figure 4. IFN- and IL-10 production by spleen cells from IRF-1 and NOD mice in the presence of Con A (panel A) or a GAD-derived peptide (panel B). Pooled spleen cells were obtained from three IRF-1 +/+ and −/− NOD mice at 30 weeks of age. The cells were cultured in the presence of Con A (panel A) or a GAD-derived peptide (panel B). An IFN-/IL-10 ratio was calculated by dividing the mean IFN- concentration by the mean IL-10 concentration in triplicate wells. Closed column, IRF-1 +/+ NOD mice; hatched column, IRF-1 −/− NOD mice; *, P<0.05 (Mann-Whitney test).
effector phases of type 1 diabetes in NOD mice [24]. IFN- production decreased and IL-10 production increased with Con A stimulation in IRF-1 −/− NOD mice, whereas both productions decreased with
GAD65-peptide stimulation. Although the reason for the difference between the cytokine responses to Con A and GAD65 peptide is unclear, IFN- production and the ratio of Th1/Th2 cytokine (IFN-/IL-10)
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productions significantly decreased in both cases, suggesting an immune deviation to the Th2 type response. The decreased production of IFN- may have protected -cells from autoimmune destruction, because various reports indicate that the development of autoimmune diabetes in NOD mice is accelerated under the predominance of Th1 cytokines [2], and because the decreased production of IFN- downregulates iNOS expression and production of NO, one of mediators of -cell destruction [20], in macrophages and -cells. Actually it has been reported that iNOS induction is impaired in IRF-1-deficient mice [6]. In addition to the decreased IFN- production, defective IFN--mediated immune responses via IRF-1 may have impaired autoimmune pathways in NOD mice. On the other hand, it has been indicated that IFN- and IFN- receptor system does not profoundly influence NOD mouse diabetes. Genetic absence of IFN- delays but does not prevent diabetes in NOD mice [25]. Lack of IFN- receptor confers resistance to cyclophosphamide-induced diabetes without affecting the natural course of diabetes development in NOD mice [26]. Although IRF-1 deficiency completely prevented diabetes in NOD mice, the influence of defect in IFN- and IFN-R system is limited [25, 26]. This discrepancy is understandable, because IRF-1, one of key molecules in host defense and immune system, is induced not only by type II IFN (IFN-) but also by type I IFNs (IFN- and IFN-) [27]. Thus, it is conceivable that multiple mechanisms mediated by IRF-1 may have prevented the development of autoimmune diabetes in IRF-1-deficient NOD mice. We have shown that the knockout of IRF-1 gene expression completely suppressed the development of autoimmune diabetes, although the exact immune mechanisms remain to be elucidated. We conclude that the signal via IRF-1 plays a pivotal role in the development of anti-islet autoimmunity in NOD mice.
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Acknowledgements
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This work was supported in part by Grants-in-Aid for Scientific Research (10671053) from the Ministry of Education, Science and Culture, Japan. The results of this study were presented in a preliminary form at the Annual Meeting of the American Diabetes Association, Chicago, June 1998.
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