- Email: [email protected]
Dopamine D1-like receptor antagonist, SCH23390, exhibits a preventive effect on diabetes mellitus that occurs naturally in NOD mice
Biochemical and Biophysical Research Communications 383 (2009) 460–463
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Dopamine D1-like receptor antagonist, SCH23390, exhibits a preventive effect on diabetes mellitus that occurs naturally in NOD mice Kumiko Hashimoto a, Tsutomu Inoue b, Takehiro Higashi a, Shin-ichiro Takei c, Takuya Awata c, Shigehiro Katayama c, Rie Takagi a, Hirokazu Okada b, Sho Matsushita a,* a
Department of Allergy and Immunology, Saitama Medical University, Saitama, 38 Morohongo, Moroyama, Saitama 350-0495, Japan Department of Nephrology, Saitama Medical University, Saitama, 38 Morohongo, Moroyama, Saitama 350-0495, Japan c Department of Endocrinology and Diabetes, Faculty of Medicine, Saitama Medical University, Saitama, 38 Morohongo, Moroyama, Saitama 350-0495, Japan b
a r t i c l e
i n f o
Article history: Received 2 April 2009 Available online 14 April 2009
Keywords: Dopamine receptor Th17 NOD D1-like antagonist IL-23 receptor
a b s t r a c t Dopamine receptors have five isoforms, termed D1–D5. The D1 and D5 receptors form the D1-like group that couples with the Gas class of G proteins, while D2, D3 and D4 form the D2-like group that couples with the Gai class of G proteins. In our previous studies, a D1-like-R antagonist, SCH23390, inhibited DC-mediated Th17 differentiation and exhibited preventive and therapeutic effects on experimental autoimmune encephalomyelitis (EAE) in mice. We herein demonstrate in the current study that in the pancreas obtained from NOD mice, islet infiltrates appear to be composed of mononuclear cells positive for IL-23R, one of the specific markers for Th17. Thereafter, NOD mice were orally administered SCH23390 from week 6 to week 26. At week 26, 67% and 25% of mice developed diabetes in the control and the SCH23390 groups, respectively (p < 0.05). A histological examination of SCH23390-treated mice exhibited a typical normal islet structure with no signs of periductal and perivascular infiltrates, whereas the islets from vehicle controls showed insulitis. In week 26, spleen cells were re-stimulated with anti-CD3 and anti-CD28 antibodies in vitro and exhibited an augmentation of IFNc induction and the suppression of IL-17 induction in the SCH23390-treated mice. These findings indicate that antagonizing D1like-R suppresses IL-17 expression, thereby leading to a decreased occurrence of NOD. Ó 2009 Elsevier Inc. All rights reserved.
Introduction The different classes of specific immune responses are driven by the biased development of antigen-specific effector CD4+T-cell subsets such as Th1, Th2 and Th17 cells that activate different components of cellular and humoral immunity. The Th17 lineage produces characteristically high levels of IL-17, and it has also been identified to represent a significant revision of the Th1– Th2 paradigm [1,2]. Moreover, Th17 cells have been reported to play not only critical roles in the immune responses to extracellular bacteria but also a pathogenetic role in autoimmunity [1,3–5]. DCs reside in an immature state in many nonlymphoid tissues such as the skin or airway mucosa which undergo frequent exposure to allergens, pathogens and chemicals. The stimulatory signal results from the ligation of TCRs by antigenic peptides presented by the MHC class II molecules on the cell surface of DCs. TCR ligation with co-stimulation allows naïve T cells to develop into effector cells, normally accompanied by a high-level * Corresponding author. Fax: +81 49 294 2274. E-mail address: [email protected] (S. Matsushita). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.04.034
expression of selective sets of cytokines. It is well-known that the balance of these cytokines and the resulting class of immune responses strongly depend on the conditions under which the DCs are primed [6]. In our previous studies, we showed that antagonizing dopamine-receptor subtypes differentially affected Th17 polarization in vitro [7,8]. Furthermore, D2-like antagonists judged to be Th17 adjuvants in vitro cause a marked deterioration of experimental autoimmune encephalomyelitis (EAE), whereas D1-like-R antagonists exhibited a marked improvement in EAE. In the present study, we show that D1-like-R antagonist SCH23390 exhibits a preventive effect on diabetes mellitus that occurs naturally in NOD mice. Materials and methods Immunohistochemical analysis of the pancreas. Paraformaldehyde-fixed, paraffin-embedded pancreas sections of 4-lm thickness were deparaffinized and rehydrated for any examinations. The sections were autoclaved (121 °C, for 15 min) with Antigen Unmasking Solution (Vector, Burlingame, CA) for antigen retrieval. The antibody used for immunohistochemistry was anti-IL-23R
K. Hashimoto et al. / Biochemical and Biophysical Research Communications 383 (2009) 460–463
(LifeSpan, Seattle, WA), which was diluted 1- to 30-fold with 1% BSA in PBS as the blocking buffer, and the sections were incubated with the primary antibody for 12 h at 37 °C. The reaction was amplified using tyramide signal amplification kit and labeled with Alexa Fluor 555 (Molecular Probe/Invitrogen, Carlsbad, CA). The negative control sections were treated as described above but rabbit IgG was used for the primary antibody. TO-PRO-3 (Molecular Probe) was used to stain the nuclei. A confocal laser scanning microscope (FV1000; Olympus, Tokyo, Japan) was employed for the data acquisition. NOD mice. The female NOD mice used in this study were obtained from CLEA Japan, Inc. (Japan). All mice were maintained under specific pathogen-free conditions. In the NOD colony, the cumulative incidence of diabetes was 50–70% in females and less than 20% in males by 26 weeks of age. The mice were orally administered with 0.3 mg/kg SCH23390 twice a week from week 6 to week 26. The mice in the control group received 50 ll PBS (n = 12 for each group). The urine sugar of NOD mice was checked every week to detect the onset of diabetes. The 26week-old mice were sacrificed using ether anesthesia in order to perform islet histology, and the spleen was obtained for the in vitro immunological assay. Pancreatic tissue specimens were fixed with 10% buffered formalin, and were embedded in paraffin. Three serial 5 lm sections were cut at intervals of 100 lm stained with hematoxylin and eosin (HE). A minimum of 20 islets from each animal was examined and the severity of insulitis was determined. The animal experiments were approved by and performed in compliance with the guidelines of the Institutional Animal Care and Use Committee. The mice were maintained under pathogen-free conditions with free access to normal mouse chow and water. Determination of cytokine responses. Spleen cells from 26week-old NOD mice (1 107/ml) were suspended in RPMI 1640 medium containing 10% FCS and were re-stimulated with anti-CD3 and anti-CD28 antibodies as described in a previous study [7]. All samples were analyzed in triplicate. The comparisons between sets of two groups were performed using Student’s two-tailed t-test, while sets of more than two groups were compared by ANOVA.
461
Results Immunohistochemical analysis We first tried to stain intracellular IL-17 by using the pancreas specimens, but we could not obtain any positive results. Thereafter, we used anti-IL-23Ra antibody to detect one of specific surface markers for Th17 cells [9]. Only the combination of autoclaving (121 °C, for 15 min) with Antigen Unmasking Solution yielded successful results. Neither proteinase treatment nor microwaving was successful (data not shown). A number of IL-23R+mononuclear cells were found in the inflamed islets of the pancreas from 21week-old NOD mice (Fig. 1), although any 15-, 17- or 19-weekold mice did not have such cells in the islets. Effect of SCH23390 on diabetes The mice were orally administered with 0.3 mg/kg SCH23390 twice a week from week 6 to week 26. The mice in the control group received 50 ll PBS. The urine sugar level was checked every week to detect onset of diabetes. The mice of the control group started to develop diabetes at 16 weeks of age, whereas the mice in the SCH23390 group developed diabetes at 20 weeks of age. At week 21, 100% and 6% of randomly selected islets of the control group and the SCH23390 group, respectively, exhibited insulitis (data not shown). At week 26, 67% and 25% of mice developed diabetes in the control and the SCH23390 groups, respectively, and these incidences were significantly different (p < 0.05) (Fig. 2A). Fig. 2B shows that the histological examination of 26-week-old SCH23390-treated mice showed a typical normal islet structure with no signs of periductal and perivascular infiltrates, whereas the islet from vehicle controls showed insulitis with some structures completely disrupted. Cytokine responses The cytokine responses were examined in week 26 (Fig. 3). The spleen cells were re-stimulated with anti-CD3 and anti-CD28 antibodies in vitro and were subjected to cytokine analysis. The aug-
Fig. 1. IL-23R+Th17 cell infiltration into the islets in the NOD mice. Pancreatic sections were autoclaved with Antigen Unmasking Solution for antigen retrieval. The sections were incubated with anti-IL-23R antibody for 12 h at 37 °C. The reaction was amplified using tyramide signal amplification kit and labeled with Alexa Fluor 555. Negative control sections were treated as described above with rabbit IgG.
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K. Hashimoto et al. / Biochemical and Biophysical Research Communications 383 (2009) 460–463
diabetes development
% 70
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60
SCH23390
50
*
40 30 20 10
*: p<0.05
0 15
20
25
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26
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SCH23390
100
100
400
400
Fig. 2. The effect of DA-R antagonist SCH23390 on NOD mice. (A) The mice were orally administered with 0.3 mg/kg SCH23390 twice a week from week 6 to 26. The mice in the control group received 50 ll PBS. The urine sugar was checked every week to detect onset of diabetes. (B) Histological examination of NOD pancreata. The mice without urinary sugar on week 26 were analyzed in both control and SCH23390 groups.
IFNγ ng/ml
IL-4 pg/ml
**
20
IL-17 ng/ml
*
**
1.8
140
1.5 100 1
10 50
0.5
0
0 control
SCH
0 control
SCH
control
SCH
Fig. 3. Spleen cell responses in NOD. Spleen cells of both groups of mice were re-stimulated with anti-CD3 and anti-CD28 Abs to be subjected to ELISA for cytokines. *p < 0.05, ** p < 0.01.
mentation of IFNc induction and the suppression of IL-17 induction were observed in the SCH23390-treated mice. Discussion Th17 cells have been described as a distinct subset of effector cells that differentiate from naïve T cells in response to IL-6 and transforming growth factor-b (TGFb) [10]. Recent studies have demonstrated that Th17 cells, rather than Th1 cells, play a pivotal role in the pathogenesis of autoimmune disease models, including EAE and collagen-induced arthritis (CIA) [11,12]. The current experiments indicate that antagonizing D1-like-R leads to a sup-
pression of IL-17 production and prevents naturally occurring diabetes in NOD mice. The effect of D1-like-R antagonist SCH23390 is not a simple non-specific immunosuppression but instead a qualitative change in the immune responses, because the suppression of IL-17 production is accompanied by the augmentation of IFNc production. The NOD mice spontaneously develop autoimmune diabetes which shares several genetic and immunopathological features with type 1 diabetes [13]. The mononuclear cells which are primarily composed of T cells infiltrate into the islets, thus resulting in the development of insulitis. In particular, CD4+T cells play important roles in initiating and propagating the autoimmune process [14].
K. Hashimoto et al. / Biochemical and Biophysical Research Communications 383 (2009) 460–463
The results obtained from the present study clearly support the novel notion that a Th17 lineage was involved in the development of NOD. Indeed, several studies by others suggest such an interpretation. Radhakrishnan, et al. reported that Th17 cells induce diabetes in rat-insulin promoter-OVA mice [15]. Diabetes induction can be accelerated by IL-23 treatment that leads to IL-17 induction [16], and IL-17 has been shown to increase the level of inducible nitric oxide (NO) synthase in pancreatic b cells, leading to the production of NO and subsequent b cell damage [17]. In the present study, islet infiltrates appear to be composed of mononuclear cells positive for IL-23R, one of the specific Th17 markers that can distinguish the Th17 population from Th1, Th2, or Tregs [9,18]. Furthermore, Spolski, et al. reported that IL-21R-knockout mice backcrossed with NOD mice, were devoid of lymphocytic infiltration into the pancreas, and only 1 of 20 animals had an elevated glucose in comparison to 60% of NOD mice that had a wild-type background [19]. Although TCR and Treg-related responses were normal, these mice had reduced Th17 cells and significantly higher levels of mRNAs encoding members of the Reg (regenerating) gene family whose transgenic expression protects against diabetes [19]. The precise mechanism of how SCH23390 inhibits IL-17 production, however, remains undetermined. In our previous studies with EAE model mice, the adoptive transfer of DCs treated with SCH23390 successfully prevented EAE [7]. Dopamine stored in DCs is released upon the interaction with naïve CD4+T cells, and increases the formation of cAMP in naïve CD4+T cells, thereby polarizing Th2 differentiation [8]. There is a possibility that SCH23390 decreases cAMP formation and subsequently decreases dopamine synthesis in Mo-DCs, thereby polarizing Th1 differentiation, and thus leading to an inhibition of IL-17 production. Alternatively, it is also likely that signaling through D1-like-R and D2-like-R affects the expression patterns of certain molecules, such as Notch ligands on DCs [20–23]. The therapeutic effect of SCH23390 on already-induced diabetes is currently under investigation. Acknowledgments This research was supported by grants from an Internal Research Grant, Saitama Medical University, the Ministry of Education, Culture, Sports, Science and Technology (Japan), and the Ministry of Health, Labor and Welfare (Japan). References [1] H. Park, Z. Li, X.O. Yang, S.H. Chang, R. Nurieva, Y.H. Wang, Y. Wang, L. Hood, Z. Zhu, Q. Tian, C. Dong, A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17, Nat. Immunol. 6 (2005) 1133–1141. [2] L.E. Harrington, R.D. Hatton, P.R. Mangan, H. Turner, T.L. Murphy, K.M. Murphy, C.T. Weaver, Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages, Nat. Immunol. 6 (2005) 1123–1132. [3] S. Nakae, A. Nambu, K. Sudo, Y. Iwakura, Suppression of immune induction of collagen-induced arthritis in IL-17-deficient mice, J. Immunol. 171 (2003) 6173–6177.
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[4] C.T. Weaver, R.D. Hatton, P.R. Mangan, L.E. Harrington, IL-17 family cytokines and the expanding diversity of effector T cell lineages, Annu. Rev. Immunol. 25 (2007) 821–852. [5] E.V. Acosta-Rodriguez, L. Rivino, J. Geginat, D. Jarrossay, M. Gattorno, A. Lanzavecchia, F. Sallusto, G. Napolitani, Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells, Nat. Immunol. 8 (2007) 639–646. [6] T. Higashi, M. Wakui, K. Nakano, K. Hashimoto, R. Takagi, Y. Tanaka, S. Matsushita, Evaluation of adjuvant activities using human antigen presenting cells in vitro, Allergol. Int. 57 (2008) 219–222. [7] K. Nakano, T. Higashi, K. Hashimoto, R. Takagi, Y. Tanaka, S. Matsushita, Antagonizing dopamine D1-like receptor inhibits Th17 cell differentiation: preventive and therapeutic effects on experimental autoimmune encephalomyelitis, Biochem. Biophys. Res. Commun. 373 (2008) 286–291. [8] K. Nakano, T. Higashi, R. Takagi, K. Hashimoto, Y. Tanaka, S. Matsushita, Dopamine released by dendritic cells polarizes Th2 differentiation, Int. Immunol., in press. [9] R. Takagi, T. Higashi, K. Hashimoto, K. Nakano, Y. Mizuno, Y. Okazaki, S. Matsushita, B cell chemoattractant CXCL13 is preferentially expressed by human Th17 cell clones, J. Immunol. 181 (2008) 186–189. [10] P.R. Mangan, L.E. Harrington, D.B. O’Quinn, W.S. Helms, D.C. Bullard, C.O. Elson, R.D. Hatton, S.M. Wahl, T.R. Schoeb, C.T. Weaver, Transforming growth factorbeta induces development of the T(H)17 lineage, Nature 441 (2006) 231–234. [11] D.J. Cua, J. Sherlock, Y. Chen, C.A. Murphy, B. Joyce, B. Seymour, L. Lucian, W. To, S. Kwan, T. Churakova, S. Zurawski, M. Wiekowski, S.A. Lira, D. Gorman, R.A. Kastelein, J.D. Sedgwick, Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain, Nature 421 (2003) 744–748. [12] C.A. Murphy, C.L. Langrish, Y. Chen, W. Blumenschein, T. McClanahan, R.A. Kastelein, J.D. Sedgwick, D.J. Cua, Divergent pro- and antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation, J. Exp. Med. 198 (2003) 1951–1957. [13] S. Makino, K. Kunimoto, Y. Muraoka, Y. Mizushima, K. Katagiri, Y. Tochino, Breeding of a non-obese, Diabetic strain of mice, Jikken Dobutsu 29 (1980) 1– 13. [14] R. Tisch, H. McDevitt, Insulin-dependent diabetes mellitus, Cell 85 (1996) 291– 297. [15] S. Radhakrishnan, R. Cabrera, E.L. Schenk, P. Nava-Parada, M.P. Bell, V.P. Van Keulen, R.J. Marler, S.J. Felts, L.R. Pease, Reprogrammed FoxP3+ T regulatory cells become IL-17+ antigen-specific autoimmune effectors in vitro and in vivo, J. Immunol. 181 (2008) 3137–3147. [16] E.P. Mensah-Brown, A. Shahin, M. Al-Shamisi, X. Wei, M.L. Lukic, IL-23 leads to diabetes induction after subdiabetogenic treatment with multiple low doses of streptozotocin, Eur. J. Immunol. 36 (2006) 216–223. [17] D. Miljkovic, I. Cvetkovic, M. Momcilovic, D. Maksimovic-Ivanic, S. StosicGrujicic, V. Trajkovic, Interleukin-17 stimulates inducible nitric oxide synthase-dependent toxicity in mouse beta cells, Cell Mol. Life Sci. 62 (2005) 2658–2668. [18] S. Matsushita, T. Higashi, Human Th17 cell clones and natural immune responses, Allergol. Int. 57 (2008) 135–140. [19] R. Spolski, M. Kashyap, C. Robinson, Z. Yu, W.J. Leonard, IL-21 signaling is critical for the development of type I diabetes in the NOD mouse, Proc. Natl. Acad. Sci. USA 105 (2008) 14028–14033. [20] M. Wakui, K. Nakano, S. Matsushita, Notch ligand mRNA levels of human APCs predict Th1/Th2-promoting activities, Biochem. Biophys. Res. Commun. 358 (2007) 596–601. [21] D. Amsen, J.M. Blander, G.R. Lee, K. Tanigaki, T. Honjo, R.A. Flavell, Instruction of distinct CD4 T helper cell fates by different notch ligands on antigenpresenting cells, Cell 117 (2004) 515–526. [22] D. Amsen, A. Antov, D. Jankovic, A. Sher, F. Radtke, A. Souabni, M. Busslinger, B. McCright, T. Gridley, R.A. Flavell, Direct regulation of gata3 expression determines the T helper differentiation potential of notch, Immunity 27 (2007) 89–99. [23] M.A. Schaller, R. Neupane, B.D. Rudd, S.L. Kunkel, L.E. Kallal, P. Lincoln, J.B. Lowe, Y. Man, N.W. Lukacs, Notch ligand Delta-like 4 regulates disease pathogenesis during respiratory viral infections by modulating Th2 cytokines, J. Exp. Med. 204 (2007) 2925–2934.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Dopamine D1-like receptor antagonist, SCH23390, exhibits a preventive effect on diabetes mellitus that occurs naturally in NOD mice Kumiko Hashimoto a, Tsutomu Inoue b, Takehiro Higashi a, Shin-ichiro Takei c, Takuya Awata c, Shigehiro Katayama c, Rie Takagi a, Hirokazu Okada b, Sho Matsushita a,* a
Department of Allergy and Immunology, Saitama Medical University, Saitama, 38 Morohongo, Moroyama, Saitama 350-0495, Japan Department of Nephrology, Saitama Medical University, Saitama, 38 Morohongo, Moroyama, Saitama 350-0495, Japan c Department of Endocrinology and Diabetes, Faculty of Medicine, Saitama Medical University, Saitama, 38 Morohongo, Moroyama, Saitama 350-0495, Japan b
a r t i c l e
i n f o
Article history: Received 2 April 2009 Available online 14 April 2009
Keywords: Dopamine receptor Th17 NOD D1-like antagonist IL-23 receptor
a b s t r a c t Dopamine receptors have five isoforms, termed D1–D5. The D1 and D5 receptors form the D1-like group that couples with the Gas class of G proteins, while D2, D3 and D4 form the D2-like group that couples with the Gai class of G proteins. In our previous studies, a D1-like-R antagonist, SCH23390, inhibited DC-mediated Th17 differentiation and exhibited preventive and therapeutic effects on experimental autoimmune encephalomyelitis (EAE) in mice. We herein demonstrate in the current study that in the pancreas obtained from NOD mice, islet infiltrates appear to be composed of mononuclear cells positive for IL-23R, one of the specific markers for Th17. Thereafter, NOD mice were orally administered SCH23390 from week 6 to week 26. At week 26, 67% and 25% of mice developed diabetes in the control and the SCH23390 groups, respectively (p < 0.05). A histological examination of SCH23390-treated mice exhibited a typical normal islet structure with no signs of periductal and perivascular infiltrates, whereas the islets from vehicle controls showed insulitis. In week 26, spleen cells were re-stimulated with anti-CD3 and anti-CD28 antibodies in vitro and exhibited an augmentation of IFNc induction and the suppression of IL-17 induction in the SCH23390-treated mice. These findings indicate that antagonizing D1like-R suppresses IL-17 expression, thereby leading to a decreased occurrence of NOD. Ó 2009 Elsevier Inc. All rights reserved.
Introduction The different classes of specific immune responses are driven by the biased development of antigen-specific effector CD4+T-cell subsets such as Th1, Th2 and Th17 cells that activate different components of cellular and humoral immunity. The Th17 lineage produces characteristically high levels of IL-17, and it has also been identified to represent a significant revision of the Th1– Th2 paradigm [1,2]. Moreover, Th17 cells have been reported to play not only critical roles in the immune responses to extracellular bacteria but also a pathogenetic role in autoimmunity [1,3–5]. DCs reside in an immature state in many nonlymphoid tissues such as the skin or airway mucosa which undergo frequent exposure to allergens, pathogens and chemicals. The stimulatory signal results from the ligation of TCRs by antigenic peptides presented by the MHC class II molecules on the cell surface of DCs. TCR ligation with co-stimulation allows naïve T cells to develop into effector cells, normally accompanied by a high-level * Corresponding author. Fax: +81 49 294 2274. E-mail address: [email protected] (S. Matsushita). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.04.034
expression of selective sets of cytokines. It is well-known that the balance of these cytokines and the resulting class of immune responses strongly depend on the conditions under which the DCs are primed [6]. In our previous studies, we showed that antagonizing dopamine-receptor subtypes differentially affected Th17 polarization in vitro [7,8]. Furthermore, D2-like antagonists judged to be Th17 adjuvants in vitro cause a marked deterioration of experimental autoimmune encephalomyelitis (EAE), whereas D1-like-R antagonists exhibited a marked improvement in EAE. In the present study, we show that D1-like-R antagonist SCH23390 exhibits a preventive effect on diabetes mellitus that occurs naturally in NOD mice. Materials and methods Immunohistochemical analysis of the pancreas. Paraformaldehyde-fixed, paraffin-embedded pancreas sections of 4-lm thickness were deparaffinized and rehydrated for any examinations. The sections were autoclaved (121 °C, for 15 min) with Antigen Unmasking Solution (Vector, Burlingame, CA) for antigen retrieval. The antibody used for immunohistochemistry was anti-IL-23R
K. Hashimoto et al. / Biochemical and Biophysical Research Communications 383 (2009) 460–463
(LifeSpan, Seattle, WA), which was diluted 1- to 30-fold with 1% BSA in PBS as the blocking buffer, and the sections were incubated with the primary antibody for 12 h at 37 °C. The reaction was amplified using tyramide signal amplification kit and labeled with Alexa Fluor 555 (Molecular Probe/Invitrogen, Carlsbad, CA). The negative control sections were treated as described above but rabbit IgG was used for the primary antibody. TO-PRO-3 (Molecular Probe) was used to stain the nuclei. A confocal laser scanning microscope (FV1000; Olympus, Tokyo, Japan) was employed for the data acquisition. NOD mice. The female NOD mice used in this study were obtained from CLEA Japan, Inc. (Japan). All mice were maintained under specific pathogen-free conditions. In the NOD colony, the cumulative incidence of diabetes was 50–70% in females and less than 20% in males by 26 weeks of age. The mice were orally administered with 0.3 mg/kg SCH23390 twice a week from week 6 to week 26. The mice in the control group received 50 ll PBS (n = 12 for each group). The urine sugar of NOD mice was checked every week to detect the onset of diabetes. The 26week-old mice were sacrificed using ether anesthesia in order to perform islet histology, and the spleen was obtained for the in vitro immunological assay. Pancreatic tissue specimens were fixed with 10% buffered formalin, and were embedded in paraffin. Three serial 5 lm sections were cut at intervals of 100 lm stained with hematoxylin and eosin (HE). A minimum of 20 islets from each animal was examined and the severity of insulitis was determined. The animal experiments were approved by and performed in compliance with the guidelines of the Institutional Animal Care and Use Committee. The mice were maintained under pathogen-free conditions with free access to normal mouse chow and water. Determination of cytokine responses. Spleen cells from 26week-old NOD mice (1 107/ml) were suspended in RPMI 1640 medium containing 10% FCS and were re-stimulated with anti-CD3 and anti-CD28 antibodies as described in a previous study [7]. All samples were analyzed in triplicate. The comparisons between sets of two groups were performed using Student’s two-tailed t-test, while sets of more than two groups were compared by ANOVA.
461
Results Immunohistochemical analysis We first tried to stain intracellular IL-17 by using the pancreas specimens, but we could not obtain any positive results. Thereafter, we used anti-IL-23Ra antibody to detect one of specific surface markers for Th17 cells [9]. Only the combination of autoclaving (121 °C, for 15 min) with Antigen Unmasking Solution yielded successful results. Neither proteinase treatment nor microwaving was successful (data not shown). A number of IL-23R+mononuclear cells were found in the inflamed islets of the pancreas from 21week-old NOD mice (Fig. 1), although any 15-, 17- or 19-weekold mice did not have such cells in the islets. Effect of SCH23390 on diabetes The mice were orally administered with 0.3 mg/kg SCH23390 twice a week from week 6 to week 26. The mice in the control group received 50 ll PBS. The urine sugar level was checked every week to detect onset of diabetes. The mice of the control group started to develop diabetes at 16 weeks of age, whereas the mice in the SCH23390 group developed diabetes at 20 weeks of age. At week 21, 100% and 6% of randomly selected islets of the control group and the SCH23390 group, respectively, exhibited insulitis (data not shown). At week 26, 67% and 25% of mice developed diabetes in the control and the SCH23390 groups, respectively, and these incidences were significantly different (p < 0.05) (Fig. 2A). Fig. 2B shows that the histological examination of 26-week-old SCH23390-treated mice showed a typical normal islet structure with no signs of periductal and perivascular infiltrates, whereas the islet from vehicle controls showed insulitis with some structures completely disrupted. Cytokine responses The cytokine responses were examined in week 26 (Fig. 3). The spleen cells were re-stimulated with anti-CD3 and anti-CD28 antibodies in vitro and were subjected to cytokine analysis. The aug-
Fig. 1. IL-23R+Th17 cell infiltration into the islets in the NOD mice. Pancreatic sections were autoclaved with Antigen Unmasking Solution for antigen retrieval. The sections were incubated with anti-IL-23R antibody for 12 h at 37 °C. The reaction was amplified using tyramide signal amplification kit and labeled with Alexa Fluor 555. Negative control sections were treated as described above with rabbit IgG.
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K. Hashimoto et al. / Biochemical and Biophysical Research Communications 383 (2009) 460–463
diabetes development
% 70
control
60
SCH23390
50
*
40 30 20 10
*: p<0.05
0 15
20
25
control
26
week
SCH23390
100
100
400
400
Fig. 2. The effect of DA-R antagonist SCH23390 on NOD mice. (A) The mice were orally administered with 0.3 mg/kg SCH23390 twice a week from week 6 to 26. The mice in the control group received 50 ll PBS. The urine sugar was checked every week to detect onset of diabetes. (B) Histological examination of NOD pancreata. The mice without urinary sugar on week 26 were analyzed in both control and SCH23390 groups.
IFNγ ng/ml
IL-4 pg/ml
**
20
IL-17 ng/ml
*
**
1.8
140
1.5 100 1
10 50
0.5
0
0 control
SCH
0 control
SCH
control
SCH
Fig. 3. Spleen cell responses in NOD. Spleen cells of both groups of mice were re-stimulated with anti-CD3 and anti-CD28 Abs to be subjected to ELISA for cytokines. *p < 0.05, ** p < 0.01.
mentation of IFNc induction and the suppression of IL-17 induction were observed in the SCH23390-treated mice. Discussion Th17 cells have been described as a distinct subset of effector cells that differentiate from naïve T cells in response to IL-6 and transforming growth factor-b (TGFb) [10]. Recent studies have demonstrated that Th17 cells, rather than Th1 cells, play a pivotal role in the pathogenesis of autoimmune disease models, including EAE and collagen-induced arthritis (CIA) [11,12]. The current experiments indicate that antagonizing D1-like-R leads to a sup-
pression of IL-17 production and prevents naturally occurring diabetes in NOD mice. The effect of D1-like-R antagonist SCH23390 is not a simple non-specific immunosuppression but instead a qualitative change in the immune responses, because the suppression of IL-17 production is accompanied by the augmentation of IFNc production. The NOD mice spontaneously develop autoimmune diabetes which shares several genetic and immunopathological features with type 1 diabetes [13]. The mononuclear cells which are primarily composed of T cells infiltrate into the islets, thus resulting in the development of insulitis. In particular, CD4+T cells play important roles in initiating and propagating the autoimmune process [14].
K. Hashimoto et al. / Biochemical and Biophysical Research Communications 383 (2009) 460–463
The results obtained from the present study clearly support the novel notion that a Th17 lineage was involved in the development of NOD. Indeed, several studies by others suggest such an interpretation. Radhakrishnan, et al. reported that Th17 cells induce diabetes in rat-insulin promoter-OVA mice [15]. Diabetes induction can be accelerated by IL-23 treatment that leads to IL-17 induction [16], and IL-17 has been shown to increase the level of inducible nitric oxide (NO) synthase in pancreatic b cells, leading to the production of NO and subsequent b cell damage [17]. In the present study, islet infiltrates appear to be composed of mononuclear cells positive for IL-23R, one of the specific Th17 markers that can distinguish the Th17 population from Th1, Th2, or Tregs [9,18]. Furthermore, Spolski, et al. reported that IL-21R-knockout mice backcrossed with NOD mice, were devoid of lymphocytic infiltration into the pancreas, and only 1 of 20 animals had an elevated glucose in comparison to 60% of NOD mice that had a wild-type background [19]. Although TCR and Treg-related responses were normal, these mice had reduced Th17 cells and significantly higher levels of mRNAs encoding members of the Reg (regenerating) gene family whose transgenic expression protects against diabetes [19]. The precise mechanism of how SCH23390 inhibits IL-17 production, however, remains undetermined. In our previous studies with EAE model mice, the adoptive transfer of DCs treated with SCH23390 successfully prevented EAE [7]. Dopamine stored in DCs is released upon the interaction with naïve CD4+T cells, and increases the formation of cAMP in naïve CD4+T cells, thereby polarizing Th2 differentiation [8]. There is a possibility that SCH23390 decreases cAMP formation and subsequently decreases dopamine synthesis in Mo-DCs, thereby polarizing Th1 differentiation, and thus leading to an inhibition of IL-17 production. Alternatively, it is also likely that signaling through D1-like-R and D2-like-R affects the expression patterns of certain molecules, such as Notch ligands on DCs [20–23]. The therapeutic effect of SCH23390 on already-induced diabetes is currently under investigation. Acknowledgments This research was supported by grants from an Internal Research Grant, Saitama Medical University, the Ministry of Education, Culture, Sports, Science and Technology (Japan), and the Ministry of Health, Labor and Welfare (Japan). References [1] H. Park, Z. Li, X.O. Yang, S.H. Chang, R. Nurieva, Y.H. Wang, Y. Wang, L. Hood, Z. Zhu, Q. Tian, C. Dong, A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17, Nat. Immunol. 6 (2005) 1133–1141. [2] L.E. Harrington, R.D. Hatton, P.R. Mangan, H. Turner, T.L. Murphy, K.M. Murphy, C.T. Weaver, Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages, Nat. Immunol. 6 (2005) 1123–1132. [3] S. Nakae, A. Nambu, K. Sudo, Y. Iwakura, Suppression of immune induction of collagen-induced arthritis in IL-17-deficient mice, J. Immunol. 171 (2003) 6173–6177.
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[4] C.T. Weaver, R.D. Hatton, P.R. Mangan, L.E. Harrington, IL-17 family cytokines and the expanding diversity of effector T cell lineages, Annu. Rev. Immunol. 25 (2007) 821–852. [5] E.V. Acosta-Rodriguez, L. Rivino, J. Geginat, D. Jarrossay, M. Gattorno, A. Lanzavecchia, F. Sallusto, G. Napolitani, Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells, Nat. Immunol. 8 (2007) 639–646. [6] T. Higashi, M. Wakui, K. Nakano, K. Hashimoto, R. Takagi, Y. Tanaka, S. Matsushita, Evaluation of adjuvant activities using human antigen presenting cells in vitro, Allergol. Int. 57 (2008) 219–222. [7] K. Nakano, T. Higashi, K. Hashimoto, R. Takagi, Y. Tanaka, S. Matsushita, Antagonizing dopamine D1-like receptor inhibits Th17 cell differentiation: preventive and therapeutic effects on experimental autoimmune encephalomyelitis, Biochem. Biophys. Res. Commun. 373 (2008) 286–291. [8] K. Nakano, T. Higashi, R. Takagi, K. Hashimoto, Y. Tanaka, S. Matsushita, Dopamine released by dendritic cells polarizes Th2 differentiation, Int. Immunol., in press. [9] R. Takagi, T. Higashi, K. Hashimoto, K. Nakano, Y. Mizuno, Y. Okazaki, S. Matsushita, B cell chemoattractant CXCL13 is preferentially expressed by human Th17 cell clones, J. Immunol. 181 (2008) 186–189. [10] P.R. Mangan, L.E. Harrington, D.B. O’Quinn, W.S. Helms, D.C. Bullard, C.O. Elson, R.D. Hatton, S.M. Wahl, T.R. Schoeb, C.T. Weaver, Transforming growth factorbeta induces development of the T(H)17 lineage, Nature 441 (2006) 231–234. [11] D.J. Cua, J. Sherlock, Y. Chen, C.A. Murphy, B. Joyce, B. Seymour, L. Lucian, W. To, S. Kwan, T. Churakova, S. Zurawski, M. Wiekowski, S.A. Lira, D. Gorman, R.A. Kastelein, J.D. Sedgwick, Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain, Nature 421 (2003) 744–748. [12] C.A. Murphy, C.L. Langrish, Y. Chen, W. Blumenschein, T. McClanahan, R.A. Kastelein, J.D. Sedgwick, D.J. Cua, Divergent pro- and antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation, J. Exp. Med. 198 (2003) 1951–1957. [13] S. Makino, K. Kunimoto, Y. Muraoka, Y. Mizushima, K. Katagiri, Y. Tochino, Breeding of a non-obese, Diabetic strain of mice, Jikken Dobutsu 29 (1980) 1– 13. [14] R. Tisch, H. McDevitt, Insulin-dependent diabetes mellitus, Cell 85 (1996) 291– 297. [15] S. Radhakrishnan, R. Cabrera, E.L. Schenk, P. Nava-Parada, M.P. Bell, V.P. Van Keulen, R.J. Marler, S.J. Felts, L.R. Pease, Reprogrammed FoxP3+ T regulatory cells become IL-17+ antigen-specific autoimmune effectors in vitro and in vivo, J. Immunol. 181 (2008) 3137–3147. [16] E.P. Mensah-Brown, A. Shahin, M. Al-Shamisi, X. Wei, M.L. Lukic, IL-23 leads to diabetes induction after subdiabetogenic treatment with multiple low doses of streptozotocin, Eur. J. Immunol. 36 (2006) 216–223. [17] D. Miljkovic, I. Cvetkovic, M. Momcilovic, D. Maksimovic-Ivanic, S. StosicGrujicic, V. Trajkovic, Interleukin-17 stimulates inducible nitric oxide synthase-dependent toxicity in mouse beta cells, Cell Mol. Life Sci. 62 (2005) 2658–2668. [18] S. Matsushita, T. Higashi, Human Th17 cell clones and natural immune responses, Allergol. Int. 57 (2008) 135–140. [19] R. Spolski, M. Kashyap, C. Robinson, Z. Yu, W.J. Leonard, IL-21 signaling is critical for the development of type I diabetes in the NOD mouse, Proc. Natl. Acad. Sci. USA 105 (2008) 14028–14033. [20] M. Wakui, K. Nakano, S. Matsushita, Notch ligand mRNA levels of human APCs predict Th1/Th2-promoting activities, Biochem. Biophys. Res. Commun. 358 (2007) 596–601. [21] D. Amsen, J.M. Blander, G.R. Lee, K. Tanigaki, T. Honjo, R.A. Flavell, Instruction of distinct CD4 T helper cell fates by different notch ligands on antigenpresenting cells, Cell 117 (2004) 515–526. [22] D. Amsen, A. Antov, D. Jankovic, A. Sher, F. Radtke, A. Souabni, M. Busslinger, B. McCright, T. Gridley, R.A. Flavell, Direct regulation of gata3 expression determines the T helper differentiation potential of notch, Immunity 27 (2007) 89–99. [23] M.A. Schaller, R. Neupane, B.D. Rudd, S.L. Kunkel, L.E. Kallal, P. Lincoln, J.B. Lowe, Y. Man, N.W. Lukacs, Notch ligand Delta-like 4 regulates disease pathogenesis during respiratory viral infections by modulating Th2 cytokines, J. Exp. Med. 204 (2007) 2925–2934.