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The specific p38 mitogen-activated protein kinase pathway inhibitor FR167653 keeps insulitis benign in nonobese diabetic mice
Life Sciences 74 (2004) 1817 – 1827 www.elsevier.com/locate/lifescie
The specific p38 mitogen-activated protein kinase pathway inhibitor FR167653 keeps insulitis benign in nonobese diabetic mice Hitoshi Ando, Seiichiro Kurita, Toshinari Takamura * Department of Endocrinology and Metabolism, Kanazawa University Graduate School of Medical Science, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8641, Japan Received 1 April 2003; accepted 9 September 2003
Abstract The p38 mitogen-activated protein kinase (MAPK) pathway is important in Th1 immunity, macrophage activation, and apoptosis. Since they may be associated with h-cell destruction during the development of type 1 diabetes, we investigated the role of the p38 MAPK pathway in female nonobese diabetic (NOD) mice. Phosphorylated p38 MAPK was observed immunohistochemically in CD4+ cells that had infiltrated into the islets and part of h-cells, increasing in proportion to the severity of insulitis. Continuous oral administration of 0.08% FR167653, a specific p38 MAPK pathway inhibitor, significantly reduced the ex vivo production of interferon-g by splenic Th1 cells without affecting interleukin-4 production by Th2 cells. FR167653 administration from 4 –30 weeks of age prevented NOD mice from developing diabetes without affecting the severity of insulitis. Treatment with FR167653 after insulitis had developed (i.e. from 10 – 30 weeks of age) also prevented diabetes, further suggesting that treatment with the p38 MAPK pathway inhibitor keeps insulitis benign in NOD mice, partly by inhibiting Th1 immunity. These findings suggest that p38 MAPK is a key mediator that switches insulitis from benign to destructive in the development of type 1 diabetes. D 2003 Elsevier Inc. All rights reserved. Keywords: p38 mitogen-activated protein kinase; Type 1 diabetes; Insulitis; Nonobese diabetic mouse; Th1
Introduction Type 1 diabetes results from selective destruction of the insulin-producing h-cells in the pancreatic islets of Langerhans. Cumulative evidence suggests that h-cells are destroyed by an * Corresponding author. Tel.: +81-76-265-2234; fax: +81-76-234-4250. E-mail address: [email protected] (T. Takamura). 0024-3205/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.09.045
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autoimmune response leading to islet inflammation, also known as insulitis, which is characterized by infiltration into the islets of lymphocytes and macrophages (Castan˜o and Eisenbarth, 1990; Rabinovitch, 2000). Insulitis without progression to significant h-cell destruction (benign insulitis), however, has been shown to occur in animal models of type 1 diabetes (Kolb, 1997). One of the factors that define benign insulitis quality is the balance between Th1 and Th2 cells that infiltrate the islets (Rabinovitch, 2000; Kolb, 1997). That is, destructive insulitis appears to be associated with a predominance of Th1 cells, whereas benign insulitis is associated with a dominance of Th2 reactivity. Mitogen-activated protein kinase (MAPK) pathways, especially the p38 MAPK pathway, have been found to play significant roles in inflammation (Herlaar and Brown, 1999). In in vitro experiments, inhibitors of the p38 MAPK pathway were shown to block interferon-g (IFN-g) production by Th1 cells without affecting interleukin (IL)-4 production by Th2 cells (Rinco´n et al., 1998; Zhang and Kaplan, 2000). In addition, in vivo inhibition of the p38 MAPK pathway in T cells by expression of dominantnegative p38 MAPK has been reported to result in selective impairment of Th1 responses (Rinco´n et al., 1998). Furthermore, in transgenic mice, activation of the p38 MAPK pathway by expression of constitutively-activated MAPK kinase 6 increased production of IFN-g (Rinco´n et al., 1998). The p38 MAPK pathway is thus believed to affect Th1, but not Th2, cells, suggesting that this pathway may be a therapeutic target for diseases in which the predominance of a Th1 immune response leads to a pathological outcome. To determine if activation of the p38 MAPK pathway in inflammatory cells infiltrating into the islets is associated with human type 1 diabetes, we have assayed activation of this pathway in the nonobese diabetic (NOD) mouse model. Administration of the specific p38 MAPK pathway inhibitor, FR167653 (1-[7-(4-fluorophenyl)-1,2,3,4-tetrahydro-8-(4-pyridyl)pyrazolo[5,1-c][1,2,4]triazin-2-yl]-2-phenylethanedione sulfate monohydrate), tested whether blocking this pathway could alter the nature of the insulitis in this model (Iwata et al., 2003; Matsuoka et al., 2002; Takahashi et al., 2001; Wada et al., 2001).
Methods Animals NOD/Shi/Jic mice were initially obtained from CLEA Japan Inc. (Tokyo, Japan) and were subsequently bred and raised at the Institute for Experimental Animals, Kanazawa University Faculty of Medicine. The mice were housed under controlled environmental conditions and had free access to food and water. In our colony, insulitis becomes noticeable in most mice at 5 weeks of age. At 30 weeks of age, the cumulative incidence of diabetes is 15% in males and 60% in females. All animal procedures were performed in accordance with the standards set forth in the Guidelines for the Care and Use of Laboratory Animals at the Takara-machi Campus of Kanazawa University. Immunohistochemical detection of phosphorylated p38 MAPK in islets Two 10–week–old and four 30–week–old female NOD mice were killed, and each pancreas was dissected out and fixed in neutralized 10% formalin. Paraffin-embedded sections were immunohisto-
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chemically assayed for phosphorylated p38 MAPK by the dextran polymer conjugate two-step visualization system (EnVision+, peroxidase, Dako, Carpinteria, CA), using rabbit anti-mouse phosphorylated p38 MAPK polyclonal antibodies (New England Biolabs, Beverly, MA). Kidneys of MRLFaslpr mice were utilized as a positive control (Iwata et al., 2003), and normal rabbit serum was utilized as a negative control. On the mirror sections corresponding to phosphorylated p38 MAPK-stained sections, CD4 or insulin was immunohistochemically stained using rat anti-mouse CD4 antibody (BD Biosciences, San Jose, CA) or guinea pig anti-porcine insulin antibody (Dako) as the primary antibodies. Mouse diets Three-week-old female NOD mice were divided into two groups and fed a regular diet with or without 0.08% FR167653 (donated by Fujisawa Pharmaceutical Co., Osaka, Japan) from 4 to 30 or from 10 to 30 weeks of age. The total FR167653 dosage estimated from diet consumption was about 110 mg/ kg body weight/day throughout the experimental period. FR167653 itself did not affect the health of mice, as seen by their dietary intake and body weight (Fig. 1), and glucose levels (data not shown). Assessment and diagnosis of diabetes Glucose levels were measured weekly (Glucocard, Aventis Pharma, Tokyo, Japan) beginning at 10 weeks of age. Mice were diagnosed as diabetic when two consecutive fed blood glucose levels were = 11.1 mmol/l. Assessment of insulitis Mice were sacrificed at 10 and 30 weeks of age. Paraffin-embedded sections were stained with hematoxylin and eosin, and the degree of insulitis was assigned to each islet. No insulitis was scored as 0; peripheral insulitis in < 10% of the total islet area was scored as 1; insulitis in 10–50% of the total
Fig. 1. Effects of FR167653 on dietary intake and body weight of NOD mice. Female NOD mice were fed a regular diet with (the dotted line and open bars) or without (the solid line and filled bars) 0.08% FR167653 from 4 to 16 weeks of age. FR167653 affected neither their body weight (lines) nor dietary intake at 5, 10, and 15 weeks of age (bars). Values are means F S.E. for 12 mice in each group.
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area was scored as 2; and insulitis in >50% of the total area was scored as 3. At least 20 islets per animal were scored in a blinded fashion. An insulitis index was calculated using the formula, sum of (number of islets in each category category)/total number of islets observed (Takamura et al., 1999). Cytokine measurements Ten–week–old mice treated with or without FR167653 from 4 weeks of age were killed and their spleens were obtained under aseptic conditions. Spleen cells were prepared by depleting red blood cells with ACK lysing buffer (0.15 mol/l NH4Cl, 1.0 mmol/l KHCO3, 0.1 mmol/l Na2EDTA, pH 7.2). The cells (1.0 108 cells/ml/well) were cultured for 48 hours in RPMI 1640 medium supplemented with 5% fetal bovine serum (Gibco BRL, Grand Island, NY), in the presence of 5 Ag/ml monoclonal antimouse CD3q antibody (R and D Systems, Minneapolis, MN) at 37jC in an atmosphere of 5% CO2/95% air. Concentrations of IL-4 and IFN-g in the culture supernatants were determined with enzyme-linked immunosorbent assay kits (Endogen, Woburn, MA), using the protocols recommended by the manufacturer. The lower limits of quantification of IL-4 and IFN-g were set at 5 and 10 pg/ml, respectively. The intra- and interassay coefficients of variation were all < 10%.
Fig. 2. Representative photomicrographs of phosphorylated p38 MAPK-positive cells in islets of female NOD mice at 10 (A, B, C) and 30 (D) weeks of age. Phosphorylated p38 MAPK was assayed immunohistochemically in paraffin-embedded sections by the dextran polymer conjugate two-step visualization system, using rabbit anti-mouse phosphorylated p38 MAPK polyclonal antibodies. Phosphorylated p38 MAPK-positive cells (arrows) increased in proportion to the degree of insulitis. Scale bar, 100 Am.
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Statistics Statistical analysis of the incidence of diabetes used the Kaplan–Meier method, and P values were determined with the Log-rank test. Differences in the insulitis indices and in the levels of cytokines between FR167653-treated and control mice were determined with the nonparametric Mann-Whitney test. Values are presented as means F S.E., and a P value of less than 0.05 was considered significant. All calculations were performed with the computer program Statview, version 5.0, for Macintosh (SAS Institute, Cary, NC).
Results To investigate whether the p38 MAPK pathway is activated in our experimental model of insulitis, the presence of phosphorylated p38 MAPK was immunohistochemically evaluated in the islets of female NOD mice. Positive staining could not be detected in intact islets without insulitis (Fig. 2A), while
Fig. 3. Immunohistochemical analyses of CD4 (B) and insulin (D) using mirror sections corresponding to phosphorylated p38 MAPK-stained sections (A and C). CD4 and insulin were stained using rat anti-mouse CD4 antibody and guinea pig antiporcine insulin antibody, respectively. Most of phosphorylated p38 MAPK-positive cells were also stained for CD4, while part of them (arrows) were stained for insulin. Scale bar, 200 Am.
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phosphorylated p38 MAPK was observed in islets with insulitis (Fig. 2B, C and D). The number of positively stained infiltrating inflammatory cells increased in proportion to the severity of insulitis. To elucidate the islet localization of phosphorylated p38 MAPK, CD4 and insulin were immunohistochemically examined using mirror sections corresponding to phosphorylated p38 MAPK-stained sections (Fig. 3). Most of phosphorylated p38 MAPK-positive cells were also stained for CD4 (Fig. 3A and B), while part of them were stained for insulin (Fig. 3C and D). Thus, p38 MAPK can be activated both in infiltrating cells and in pancreatic h-cells in the development of diabetes. Since the p38 MAPK pathway has been reported to be related to Th1 immunity (Rinco´n et al., 1998; Zhang and Kaplan, 2000), we examined whether feeding female NOD mice with the p38 MAPK inhibitor FR167653 for 6 weeks reduced ex vivo production of IFN-g or IL-4 by their anti-mouse CD3q antibody stimulated spleen cells. While IL-4 production did not differ between FR167653-treated and control mice (101 F 6 vs. 120 F 27 pg/ml; Fig. 4B), the production of IFN-g in FR167653-treated mice was
Fig. 4. Effects of FR167653 on spleen cell production of IFN-g (A) and IL-4 (B). Ten – week-old mice treated with (open circle) or without (closed circle) 0.08% FR167653 from 4 weeks of age were sacrificed, and their spleen cells were obtained. The cells (1.0 108/well) were stimulated with anti-mouse CD3q antibody for 48 h, and IFN-g and IL-4 in the supernatants were assessed by enzyme-linked immunosorbent assays. Values are means + S.E. for 4 mice in each group. * P < 0.05.
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significantly reduced compared with that in control mice (369 F 33 vs. 585 F 42 ng/ml; P < 0.05; Fig. 4A), suggesting that FR167653 suppresses Th1 reactivity in vivo. To further examine the effect of FR167653 on the development of diabetes, we fed female NOD mice with this compound from 4 to 30 weeks of age. While 10 of 16 control mice (63%) had developed diabetes by 30 weeks of age, only one of the 16 mice treated with FR167653 (6%) became diabetic ( P < 0.001; Fig. 5A). Histologically, insulitis in female NOD mice occurred before 10 weeks of age and was further developed at 30 weeks of age (Table 1). The administration of FR167653, however, had no effect on the severity of insulitis, as determined by insulitis indices, at either 10 or 30 weeks of age (Table 1). This finding suggests that FR167653 does not prevent NOD mice from developing type 1 diabetes by inhibiting the development of insulitis, but rather by keeping insulitis benign. To confirm this, we tested
Fig. 5. Effects of FR167653 on development of spontaneous diabetes in NOD mice. Female NOD mice were fed a regular diet with (open circle, n = 16) or without (closed circle, n = 16) 0.08% FR167653, from 4 to 30 weeks of age (A) or from 10 to 30 weeks of age (B). The prevalence of diabetes between groups with and without FR167653 was statistically significant (A, P < 0.001; B, P = 0.010).
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Table 1 Severity of insulitis in FR167653-treated NOD mice Treatment group
Age (weeks)
n
Insulitis index
Control FR167653 Control FR167653
10 10 30 30
8 8 6 6
1.03 0.95 1.58 2.09
F F F F
0.22 0.14 0.46 0.42
Data are means F S.E. None of the differences between treated and control groups at 10 and 30 weeks of age are significant.
whether treatment with FR167653 after insulitis had occurred could prevent diabetes. As expected, feeding with FR167653 from 10 to 30 weeks of age (i.e. after the onset of insulitis) reduced the prevalence of diabetes, from 56 to 13% ( P = 0.010; Fig. 5B), without affecting the development of insulitis (data not shown).
Discussion A series of pyridinyl imidazole compounds, including FR167653, constitute a novel class of compounds that specifically inhibit the p38 MAPK pathway both in vitro and in vivo (Herlaar and Brown, 1999; Iwata et al., 2003; Jackson et al., 1998; Matsuoka et al., 2002; Takahashi et al., 2001; Wada et al., 2001). These inhibitors compete with ATP at the ATP-binding site of p38 MAPK (Frantz et al., 1998; Gum et al., 1998). FR167653 dose dependently inhibits p38 MAPK activity without affecting the activities of other kinases, such as extracellular signal-regulated kinase, c-Jun N-terminal kinase, protein kinase A, protein kinase C, and cyclooxygenases (Takahashi et al., 2001). Because it has been shown that SB203580, another p38 MAPK pathway inhibitor, inihibits not only p38 MAPK activity but also the activities of c-Jun N-terminal kinase (Ammendrup et al., 2000) and cyclooxygenases (Takahashi et al., 2001), FR167653 appears to have especially high selectivity. Continuous treatment of mice with FR167653 actually inhibits in vivo activation of p38 MAPK (Iwata et al., 2003; Matsuoka et al., 2002). The results presented here clearly demonstrate that FR167653 prevents NOD mice from developing diabetes, but without inhibiting insulitis development. These findings suggest that the p38 MAPK pathway may be important for the development of diabetes, especially in the h-cell destruction that occurs following the development of insulitis. In addition to its involvement in the production of Th1 cytokines, the p38 MAPK pathway is involved in the in vivo production of macrophage-derived cytokines, such as IL-1h and tumor necrosis factor-a (TNF-a) (Jackson et al., 1998). These cytokines have been shown to be cytotoxic to pancreatic islets in vitro (Rabinovitch, 2000), and TNF-a has been reported to contribute to the development of both diabetes and insulitis (Hunger et al., 1997; Yang et al., 1994). In addition, macrophages are thought to be important in the initiation of insulitis in cyclophosphamide-treated NOD mice (Charlton et al., 1997; Lee et al., 1988). Using another model of type 1 diabetes, in which mice are treated with multiple low-doses of streptozotocin, we have shown that a thiazolidinedione compound, pioglitazone, prevents these animals from developing insulitis and diabetes, at least partly by inhibiting macrophage activation (Takamura et al., 1999). Thiazolidine-
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dione is a direct ligand for peroxisome proliferator-activated receptor-g, which reportedly inhibits macrophage activation, including the production of cytokines such as IL-1h and TNF-a, partly by antagonizing the activity of nuclear factor-nB (Jiang et al., 1998; Ricote et al., 1998). Since p38 MAPK activation may contribute to the activation of nuclear factor-nB (Herlaar and Brown, 1999; Saccani et al., 2002) and since the p38 MAPK pathway inhibitor FR167653 inhibits IL-1h and TNF-a production in the same manner as thiazolidinedione does (Yamamoto et al., 1996), we thought it possible that FR167653 may prevent NOD mice from developing insulitis. However, FR167653 did not prevent insulitis development. Although it is probable that inhibitory effects of the compound on IL-1h and TNF-a production play a role in the prevention of diabetes, these effects might not be strong enough to prevent insulitis development. The p38 MAPK pathway also contributes to the development of apoptosis (Herlaar and Brown, 1999). For example, p38 MAPK is reported to be required for Fas ligand expression in T-cells (Hsu et al., 1999). In addition, overexpression of the MAPK kinases specific for the activation of p38 MAPK or the c-Jun NH2-terminal kinase can result in apoptosis in T cells and fibroblasts (Huang et al., 1997; Johnson et al., 1996). In other systems, however, p38 MAPK activity is not necessary for apoptosis and even inhibits its development (Huang et al., 1997; Zechner et al., 1998). Thus, the role of p38 MAPK in apoptosis remains controversial (Obata et al., 2000). Interestingly, in NOD mice, interactions between Fas and the Fas ligand contribute to insulitis development as well as to h-cell destruction (Nakayama et al., 2002). Moreover, recent studies (Ammendrup et al., 2000; Pavlovic et al., 2000) have shown that inhibition of p38 MAPK does not reduce cytokine-induced apoptosis in h-cells in vitro. Therefore, it is not likely that the FR167653-induced prevention of diabetes development in NOD mice was mainly due to the direct inhibition of Fas ligand expression in T-cells or to the direct blocking of apoptosis in h-cells. However, since we detected phosphorylated p38 MAPK also in part of h-cells, it is probable that FR167653 has some direct influence on h-cells. Evidence has accumulated that Th1 cells play important roles in destructive insulitis. IFN-g and TNF-h have been shown to be cytotoxic to pancreatic islet cells in vitro (Rabinovitch, 1994, 2000), and in vivo transgenic overexpression of IFN-g or IL-2 by h-cells in non-diabetes-prone mice was found to induce h-cell destructive insulitis and autoimmune diabetes (Rabinovitch, 2000; Sarvetnick et al., 1988, 1990). Moreover, correlations have been observed between the expression of IFN-g by Th1 cells and destructive insulitis in both NOD mice and human type 1 diabetes, whereas expression of IL-4 by Th2 cells tended to correlate with benign insulitis (Rabinovitch, 2000). These findings suggest that the p38 MAPK pathway inhibitor kept insulitis benign at least partly by inhibiting Th1 immunity. However, because FR167653 might affect not only Th1 immunity but also macrophage activation and directly h-cell death, further studies are needed to clarify the precise mechanisms. While the progression from benign to destructive insulitis might be controlled at the level of the innate immune system and/or antigen-presenting cells (Kolb, 1997), the results presented here also suggest that the quality of insulitis can be modulated even after insulitis has developed. To summarize, our data provide evidence for a critical role of the p38 MAPK pathway in the process of h-cell destruction in NOD mice. The p38 MAPK appears to be a key mediator that switches insulitis from benign to destructive. These findings suggest that p38 MAPK pathway may be an attractive therapeutic target for the prevention of h-cell destruction in prediabetic patients with hcell autoimmunity.
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The specific p38 mitogen-activated protein kinase pathway inhibitor FR167653 keeps insulitis benign in nonobese diabetic mice Hitoshi Ando, Seiichiro Kurita, Toshinari Takamura * Department of Endocrinology and Metabolism, Kanazawa University Graduate School of Medical Science, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8641, Japan Received 1 April 2003; accepted 9 September 2003
Abstract The p38 mitogen-activated protein kinase (MAPK) pathway is important in Th1 immunity, macrophage activation, and apoptosis. Since they may be associated with h-cell destruction during the development of type 1 diabetes, we investigated the role of the p38 MAPK pathway in female nonobese diabetic (NOD) mice. Phosphorylated p38 MAPK was observed immunohistochemically in CD4+ cells that had infiltrated into the islets and part of h-cells, increasing in proportion to the severity of insulitis. Continuous oral administration of 0.08% FR167653, a specific p38 MAPK pathway inhibitor, significantly reduced the ex vivo production of interferon-g by splenic Th1 cells without affecting interleukin-4 production by Th2 cells. FR167653 administration from 4 –30 weeks of age prevented NOD mice from developing diabetes without affecting the severity of insulitis. Treatment with FR167653 after insulitis had developed (i.e. from 10 – 30 weeks of age) also prevented diabetes, further suggesting that treatment with the p38 MAPK pathway inhibitor keeps insulitis benign in NOD mice, partly by inhibiting Th1 immunity. These findings suggest that p38 MAPK is a key mediator that switches insulitis from benign to destructive in the development of type 1 diabetes. D 2003 Elsevier Inc. All rights reserved. Keywords: p38 mitogen-activated protein kinase; Type 1 diabetes; Insulitis; Nonobese diabetic mouse; Th1
Introduction Type 1 diabetes results from selective destruction of the insulin-producing h-cells in the pancreatic islets of Langerhans. Cumulative evidence suggests that h-cells are destroyed by an * Corresponding author. Tel.: +81-76-265-2234; fax: +81-76-234-4250. E-mail address: [email protected] (T. Takamura). 0024-3205/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.09.045
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autoimmune response leading to islet inflammation, also known as insulitis, which is characterized by infiltration into the islets of lymphocytes and macrophages (Castan˜o and Eisenbarth, 1990; Rabinovitch, 2000). Insulitis without progression to significant h-cell destruction (benign insulitis), however, has been shown to occur in animal models of type 1 diabetes (Kolb, 1997). One of the factors that define benign insulitis quality is the balance between Th1 and Th2 cells that infiltrate the islets (Rabinovitch, 2000; Kolb, 1997). That is, destructive insulitis appears to be associated with a predominance of Th1 cells, whereas benign insulitis is associated with a dominance of Th2 reactivity. Mitogen-activated protein kinase (MAPK) pathways, especially the p38 MAPK pathway, have been found to play significant roles in inflammation (Herlaar and Brown, 1999). In in vitro experiments, inhibitors of the p38 MAPK pathway were shown to block interferon-g (IFN-g) production by Th1 cells without affecting interleukin (IL)-4 production by Th2 cells (Rinco´n et al., 1998; Zhang and Kaplan, 2000). In addition, in vivo inhibition of the p38 MAPK pathway in T cells by expression of dominantnegative p38 MAPK has been reported to result in selective impairment of Th1 responses (Rinco´n et al., 1998). Furthermore, in transgenic mice, activation of the p38 MAPK pathway by expression of constitutively-activated MAPK kinase 6 increased production of IFN-g (Rinco´n et al., 1998). The p38 MAPK pathway is thus believed to affect Th1, but not Th2, cells, suggesting that this pathway may be a therapeutic target for diseases in which the predominance of a Th1 immune response leads to a pathological outcome. To determine if activation of the p38 MAPK pathway in inflammatory cells infiltrating into the islets is associated with human type 1 diabetes, we have assayed activation of this pathway in the nonobese diabetic (NOD) mouse model. Administration of the specific p38 MAPK pathway inhibitor, FR167653 (1-[7-(4-fluorophenyl)-1,2,3,4-tetrahydro-8-(4-pyridyl)pyrazolo[5,1-c][1,2,4]triazin-2-yl]-2-phenylethanedione sulfate monohydrate), tested whether blocking this pathway could alter the nature of the insulitis in this model (Iwata et al., 2003; Matsuoka et al., 2002; Takahashi et al., 2001; Wada et al., 2001).
Methods Animals NOD/Shi/Jic mice were initially obtained from CLEA Japan Inc. (Tokyo, Japan) and were subsequently bred and raised at the Institute for Experimental Animals, Kanazawa University Faculty of Medicine. The mice were housed under controlled environmental conditions and had free access to food and water. In our colony, insulitis becomes noticeable in most mice at 5 weeks of age. At 30 weeks of age, the cumulative incidence of diabetes is 15% in males and 60% in females. All animal procedures were performed in accordance with the standards set forth in the Guidelines for the Care and Use of Laboratory Animals at the Takara-machi Campus of Kanazawa University. Immunohistochemical detection of phosphorylated p38 MAPK in islets Two 10–week–old and four 30–week–old female NOD mice were killed, and each pancreas was dissected out and fixed in neutralized 10% formalin. Paraffin-embedded sections were immunohisto-
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chemically assayed for phosphorylated p38 MAPK by the dextran polymer conjugate two-step visualization system (EnVision+, peroxidase, Dako, Carpinteria, CA), using rabbit anti-mouse phosphorylated p38 MAPK polyclonal antibodies (New England Biolabs, Beverly, MA). Kidneys of MRLFaslpr mice were utilized as a positive control (Iwata et al., 2003), and normal rabbit serum was utilized as a negative control. On the mirror sections corresponding to phosphorylated p38 MAPK-stained sections, CD4 or insulin was immunohistochemically stained using rat anti-mouse CD4 antibody (BD Biosciences, San Jose, CA) or guinea pig anti-porcine insulin antibody (Dako) as the primary antibodies. Mouse diets Three-week-old female NOD mice were divided into two groups and fed a regular diet with or without 0.08% FR167653 (donated by Fujisawa Pharmaceutical Co., Osaka, Japan) from 4 to 30 or from 10 to 30 weeks of age. The total FR167653 dosage estimated from diet consumption was about 110 mg/ kg body weight/day throughout the experimental period. FR167653 itself did not affect the health of mice, as seen by their dietary intake and body weight (Fig. 1), and glucose levels (data not shown). Assessment and diagnosis of diabetes Glucose levels were measured weekly (Glucocard, Aventis Pharma, Tokyo, Japan) beginning at 10 weeks of age. Mice were diagnosed as diabetic when two consecutive fed blood glucose levels were = 11.1 mmol/l. Assessment of insulitis Mice were sacrificed at 10 and 30 weeks of age. Paraffin-embedded sections were stained with hematoxylin and eosin, and the degree of insulitis was assigned to each islet. No insulitis was scored as 0; peripheral insulitis in < 10% of the total islet area was scored as 1; insulitis in 10–50% of the total
Fig. 1. Effects of FR167653 on dietary intake and body weight of NOD mice. Female NOD mice were fed a regular diet with (the dotted line and open bars) or without (the solid line and filled bars) 0.08% FR167653 from 4 to 16 weeks of age. FR167653 affected neither their body weight (lines) nor dietary intake at 5, 10, and 15 weeks of age (bars). Values are means F S.E. for 12 mice in each group.
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area was scored as 2; and insulitis in >50% of the total area was scored as 3. At least 20 islets per animal were scored in a blinded fashion. An insulitis index was calculated using the formula, sum of (number of islets in each category category)/total number of islets observed (Takamura et al., 1999). Cytokine measurements Ten–week–old mice treated with or without FR167653 from 4 weeks of age were killed and their spleens were obtained under aseptic conditions. Spleen cells were prepared by depleting red blood cells with ACK lysing buffer (0.15 mol/l NH4Cl, 1.0 mmol/l KHCO3, 0.1 mmol/l Na2EDTA, pH 7.2). The cells (1.0 108 cells/ml/well) were cultured for 48 hours in RPMI 1640 medium supplemented with 5% fetal bovine serum (Gibco BRL, Grand Island, NY), in the presence of 5 Ag/ml monoclonal antimouse CD3q antibody (R and D Systems, Minneapolis, MN) at 37jC in an atmosphere of 5% CO2/95% air. Concentrations of IL-4 and IFN-g in the culture supernatants were determined with enzyme-linked immunosorbent assay kits (Endogen, Woburn, MA), using the protocols recommended by the manufacturer. The lower limits of quantification of IL-4 and IFN-g were set at 5 and 10 pg/ml, respectively. The intra- and interassay coefficients of variation were all < 10%.
Fig. 2. Representative photomicrographs of phosphorylated p38 MAPK-positive cells in islets of female NOD mice at 10 (A, B, C) and 30 (D) weeks of age. Phosphorylated p38 MAPK was assayed immunohistochemically in paraffin-embedded sections by the dextran polymer conjugate two-step visualization system, using rabbit anti-mouse phosphorylated p38 MAPK polyclonal antibodies. Phosphorylated p38 MAPK-positive cells (arrows) increased in proportion to the degree of insulitis. Scale bar, 100 Am.
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Statistics Statistical analysis of the incidence of diabetes used the Kaplan–Meier method, and P values were determined with the Log-rank test. Differences in the insulitis indices and in the levels of cytokines between FR167653-treated and control mice were determined with the nonparametric Mann-Whitney test. Values are presented as means F S.E., and a P value of less than 0.05 was considered significant. All calculations were performed with the computer program Statview, version 5.0, for Macintosh (SAS Institute, Cary, NC).
Results To investigate whether the p38 MAPK pathway is activated in our experimental model of insulitis, the presence of phosphorylated p38 MAPK was immunohistochemically evaluated in the islets of female NOD mice. Positive staining could not be detected in intact islets without insulitis (Fig. 2A), while
Fig. 3. Immunohistochemical analyses of CD4 (B) and insulin (D) using mirror sections corresponding to phosphorylated p38 MAPK-stained sections (A and C). CD4 and insulin were stained using rat anti-mouse CD4 antibody and guinea pig antiporcine insulin antibody, respectively. Most of phosphorylated p38 MAPK-positive cells were also stained for CD4, while part of them (arrows) were stained for insulin. Scale bar, 200 Am.
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phosphorylated p38 MAPK was observed in islets with insulitis (Fig. 2B, C and D). The number of positively stained infiltrating inflammatory cells increased in proportion to the severity of insulitis. To elucidate the islet localization of phosphorylated p38 MAPK, CD4 and insulin were immunohistochemically examined using mirror sections corresponding to phosphorylated p38 MAPK-stained sections (Fig. 3). Most of phosphorylated p38 MAPK-positive cells were also stained for CD4 (Fig. 3A and B), while part of them were stained for insulin (Fig. 3C and D). Thus, p38 MAPK can be activated both in infiltrating cells and in pancreatic h-cells in the development of diabetes. Since the p38 MAPK pathway has been reported to be related to Th1 immunity (Rinco´n et al., 1998; Zhang and Kaplan, 2000), we examined whether feeding female NOD mice with the p38 MAPK inhibitor FR167653 for 6 weeks reduced ex vivo production of IFN-g or IL-4 by their anti-mouse CD3q antibody stimulated spleen cells. While IL-4 production did not differ between FR167653-treated and control mice (101 F 6 vs. 120 F 27 pg/ml; Fig. 4B), the production of IFN-g in FR167653-treated mice was
Fig. 4. Effects of FR167653 on spleen cell production of IFN-g (A) and IL-4 (B). Ten – week-old mice treated with (open circle) or without (closed circle) 0.08% FR167653 from 4 weeks of age were sacrificed, and their spleen cells were obtained. The cells (1.0 108/well) were stimulated with anti-mouse CD3q antibody for 48 h, and IFN-g and IL-4 in the supernatants were assessed by enzyme-linked immunosorbent assays. Values are means + S.E. for 4 mice in each group. * P < 0.05.
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significantly reduced compared with that in control mice (369 F 33 vs. 585 F 42 ng/ml; P < 0.05; Fig. 4A), suggesting that FR167653 suppresses Th1 reactivity in vivo. To further examine the effect of FR167653 on the development of diabetes, we fed female NOD mice with this compound from 4 to 30 weeks of age. While 10 of 16 control mice (63%) had developed diabetes by 30 weeks of age, only one of the 16 mice treated with FR167653 (6%) became diabetic ( P < 0.001; Fig. 5A). Histologically, insulitis in female NOD mice occurred before 10 weeks of age and was further developed at 30 weeks of age (Table 1). The administration of FR167653, however, had no effect on the severity of insulitis, as determined by insulitis indices, at either 10 or 30 weeks of age (Table 1). This finding suggests that FR167653 does not prevent NOD mice from developing type 1 diabetes by inhibiting the development of insulitis, but rather by keeping insulitis benign. To confirm this, we tested
Fig. 5. Effects of FR167653 on development of spontaneous diabetes in NOD mice. Female NOD mice were fed a regular diet with (open circle, n = 16) or without (closed circle, n = 16) 0.08% FR167653, from 4 to 30 weeks of age (A) or from 10 to 30 weeks of age (B). The prevalence of diabetes between groups with and without FR167653 was statistically significant (A, P < 0.001; B, P = 0.010).
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Table 1 Severity of insulitis in FR167653-treated NOD mice Treatment group
Age (weeks)
n
Insulitis index
Control FR167653 Control FR167653
10 10 30 30
8 8 6 6
1.03 0.95 1.58 2.09
F F F F
0.22 0.14 0.46 0.42
Data are means F S.E. None of the differences between treated and control groups at 10 and 30 weeks of age are significant.
whether treatment with FR167653 after insulitis had occurred could prevent diabetes. As expected, feeding with FR167653 from 10 to 30 weeks of age (i.e. after the onset of insulitis) reduced the prevalence of diabetes, from 56 to 13% ( P = 0.010; Fig. 5B), without affecting the development of insulitis (data not shown).
Discussion A series of pyridinyl imidazole compounds, including FR167653, constitute a novel class of compounds that specifically inhibit the p38 MAPK pathway both in vitro and in vivo (Herlaar and Brown, 1999; Iwata et al., 2003; Jackson et al., 1998; Matsuoka et al., 2002; Takahashi et al., 2001; Wada et al., 2001). These inhibitors compete with ATP at the ATP-binding site of p38 MAPK (Frantz et al., 1998; Gum et al., 1998). FR167653 dose dependently inhibits p38 MAPK activity without affecting the activities of other kinases, such as extracellular signal-regulated kinase, c-Jun N-terminal kinase, protein kinase A, protein kinase C, and cyclooxygenases (Takahashi et al., 2001). Because it has been shown that SB203580, another p38 MAPK pathway inhibitor, inihibits not only p38 MAPK activity but also the activities of c-Jun N-terminal kinase (Ammendrup et al., 2000) and cyclooxygenases (Takahashi et al., 2001), FR167653 appears to have especially high selectivity. Continuous treatment of mice with FR167653 actually inhibits in vivo activation of p38 MAPK (Iwata et al., 2003; Matsuoka et al., 2002). The results presented here clearly demonstrate that FR167653 prevents NOD mice from developing diabetes, but without inhibiting insulitis development. These findings suggest that the p38 MAPK pathway may be important for the development of diabetes, especially in the h-cell destruction that occurs following the development of insulitis. In addition to its involvement in the production of Th1 cytokines, the p38 MAPK pathway is involved in the in vivo production of macrophage-derived cytokines, such as IL-1h and tumor necrosis factor-a (TNF-a) (Jackson et al., 1998). These cytokines have been shown to be cytotoxic to pancreatic islets in vitro (Rabinovitch, 2000), and TNF-a has been reported to contribute to the development of both diabetes and insulitis (Hunger et al., 1997; Yang et al., 1994). In addition, macrophages are thought to be important in the initiation of insulitis in cyclophosphamide-treated NOD mice (Charlton et al., 1997; Lee et al., 1988). Using another model of type 1 diabetes, in which mice are treated with multiple low-doses of streptozotocin, we have shown that a thiazolidinedione compound, pioglitazone, prevents these animals from developing insulitis and diabetes, at least partly by inhibiting macrophage activation (Takamura et al., 1999). Thiazolidine-
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dione is a direct ligand for peroxisome proliferator-activated receptor-g, which reportedly inhibits macrophage activation, including the production of cytokines such as IL-1h and TNF-a, partly by antagonizing the activity of nuclear factor-nB (Jiang et al., 1998; Ricote et al., 1998). Since p38 MAPK activation may contribute to the activation of nuclear factor-nB (Herlaar and Brown, 1999; Saccani et al., 2002) and since the p38 MAPK pathway inhibitor FR167653 inhibits IL-1h and TNF-a production in the same manner as thiazolidinedione does (Yamamoto et al., 1996), we thought it possible that FR167653 may prevent NOD mice from developing insulitis. However, FR167653 did not prevent insulitis development. Although it is probable that inhibitory effects of the compound on IL-1h and TNF-a production play a role in the prevention of diabetes, these effects might not be strong enough to prevent insulitis development. The p38 MAPK pathway also contributes to the development of apoptosis (Herlaar and Brown, 1999). For example, p38 MAPK is reported to be required for Fas ligand expression in T-cells (Hsu et al., 1999). In addition, overexpression of the MAPK kinases specific for the activation of p38 MAPK or the c-Jun NH2-terminal kinase can result in apoptosis in T cells and fibroblasts (Huang et al., 1997; Johnson et al., 1996). In other systems, however, p38 MAPK activity is not necessary for apoptosis and even inhibits its development (Huang et al., 1997; Zechner et al., 1998). Thus, the role of p38 MAPK in apoptosis remains controversial (Obata et al., 2000). Interestingly, in NOD mice, interactions between Fas and the Fas ligand contribute to insulitis development as well as to h-cell destruction (Nakayama et al., 2002). Moreover, recent studies (Ammendrup et al., 2000; Pavlovic et al., 2000) have shown that inhibition of p38 MAPK does not reduce cytokine-induced apoptosis in h-cells in vitro. Therefore, it is not likely that the FR167653-induced prevention of diabetes development in NOD mice was mainly due to the direct inhibition of Fas ligand expression in T-cells or to the direct blocking of apoptosis in h-cells. However, since we detected phosphorylated p38 MAPK also in part of h-cells, it is probable that FR167653 has some direct influence on h-cells. Evidence has accumulated that Th1 cells play important roles in destructive insulitis. IFN-g and TNF-h have been shown to be cytotoxic to pancreatic islet cells in vitro (Rabinovitch, 1994, 2000), and in vivo transgenic overexpression of IFN-g or IL-2 by h-cells in non-diabetes-prone mice was found to induce h-cell destructive insulitis and autoimmune diabetes (Rabinovitch, 2000; Sarvetnick et al., 1988, 1990). Moreover, correlations have been observed between the expression of IFN-g by Th1 cells and destructive insulitis in both NOD mice and human type 1 diabetes, whereas expression of IL-4 by Th2 cells tended to correlate with benign insulitis (Rabinovitch, 2000). These findings suggest that the p38 MAPK pathway inhibitor kept insulitis benign at least partly by inhibiting Th1 immunity. However, because FR167653 might affect not only Th1 immunity but also macrophage activation and directly h-cell death, further studies are needed to clarify the precise mechanisms. While the progression from benign to destructive insulitis might be controlled at the level of the innate immune system and/or antigen-presenting cells (Kolb, 1997), the results presented here also suggest that the quality of insulitis can be modulated even after insulitis has developed. To summarize, our data provide evidence for a critical role of the p38 MAPK pathway in the process of h-cell destruction in NOD mice. The p38 MAPK appears to be a key mediator that switches insulitis from benign to destructive. These findings suggest that p38 MAPK pathway may be an attractive therapeutic target for the prevention of h-cell destruction in prediabetic patients with hcell autoimmunity.
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