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IL-10 Deficiency Does Not Inhibit Insulitis and Accelerates Cyclophosphamide-Induced Diabetes in the Nonobese Diabetic Mouse
Cellular Immunology 202, 97–102 (2000) doi:10.1006/cimm.2000.1658, available online at http://www.idealibrary.com on
IL-10 Deficiency Does Not Inhibit Insulitis and Accelerates Cyclophosphamide-Induced Diabetes in the Nonobese Diabetic Mouse Balaji Balasa,* ,1 Kurt Van Gunst,* Nadja Jung,* Jonathan D. Katz,† and Nora Sarvetnick* *Department of Immunology, The Scripps Research Institute, La Jolla, California 92037; and †Department of Pathology, Center for Immunology, Washington University School of Medicine, St. Louis, Missouri 63110 Received February 24, 2000; accepted April 20, 2000
Cytokines produced by T cells and antigen-presenting cells (APC) modulate the immunopathogenesis of autoimmune diabetes. Understanding the role of cytokines will provide insights into the development of immunotherapeutic reagents for intervention in autoimmune diabetes. We and others have been focusing research on understanding the role of cytokines, in particular Th2 cytokine IL-10, in autoimmune diabetes. Work from our laboratory has shown that IL-10 is required for the development of diabetes because neutralization of IL-10 with antibodies effectively blocked insulitis development (4). Transgenic expression of IL-10 in the pancreatic islets of NOD mice accelerated their diabetes (5–7). These findings imply that IL-10 is actually an immunostimulatory factor in IDDM of the NOD mouse. To further explore the role of IL-10 in autoimmune diabetes, we introduced IL-10 gene disruption in NOD mice. We found that IL-10 deficiency does not inhibit insulitis. Splenocytes from IL-10-deficient NOD mice readily proliferated against glutamic acid decarboxylase 65 (GAD65). Importantly, IL-10-deficient NOD mice developed accelerated diabetes following cyclophosphamide (CYP) injection. These results demonstrate that IL-10 is not required for autoimmune diabetes.
IL-10 exterts profound immunostimulatory and immunoinhibitory effects. To explore the role of IL-10 in autoimmune diabetes of nonobese diabetic (NOD) mice, we generated IL-10-deficient NOD mice. In contrast to our previous results with neutralizing antibodies to IL-10, IL-10-deficient NOD mice developed insulitis and their splenocytes readily responded to islet antigen glutamic acid decarboxylase 65. IL-10deficient NOD mice did not develop accelerated spontaneous diabetes. On the other hand, IL-10-deficient NOD mice developed accelerated disease following cyclophosphamide (CYP) injection. These findings demonstrate that IL-10 is dispensable for autoimmune diabetes. IL-10’s absence fails to accelerate endogenous diabetes but potentiates CYP-induced diabetes. © 2000 Academic Press
Key Words: IL-10; insulitis; diabetes; NOD.
INTRODUCTION Insulin-dependent diabetes mellitus (IDDM) 2 is an autoimmune disease characterized by the loss of insulin-producing pancreatic  cells (1). The nonobese diabetic (NOD) mouse has been used as an animal model for IDDM in human beings (1). The autoimmune diabetes of the NOD mouse is a T-cell-mediated disease (2, 3). In its early and clinically silent phase, T cells and other immune cells infiltrate the islets, causing a progressive loss of  cells. When a majority of  cells have disappeared, the lack of insulin secretion leads to a failure of blood glucose (BG) homeostasis and diabetes.
MATERIALS AND METHODS Mice NOD/shi mice, 4 to 5 weeks old or 10 to 11 weeks old, were purchased from the rodent breeding colony at The Scripps Research Institute (La Jolla, CA). NOD-scid/ scid mice were also purchased from the rodent-breeding colony at The Scripps Research Institute.
1
B.B. is supported by postdoctoral fellowships from the Juvenile Diabetes Foundation International (JDFI) and the Myasthenia Gravis Foundation of America, Inc. This work was supported by a Diabetes Interdisciplinary Research Program grant from the JDFI (to N.S.). This is manuscript no. 12992-IMM. 2 Abbreviations used: IL-10, interleukin-10; BG, blood glucose; CYP, cyclophosphamide; GAD65, glutamic acid decarboxylase 65; IDDM, insulin-dependent diabetes mellitus; KO, knockout; NOD, nonobese diabetic; Ab, antibody.
Generation of IL-10-Deficient (⫺/⫺) Mice IL-10-deficient (⫺/⫺) C57BL/6 mice (8) (kindly provided by Dr. Ralph Kuhn and Werner Muller, Institute for Genetics, University of Cologne, Federal Republic of Germany) that were backcrossed to NOD mice for 9 97
0008-8749/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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generations (by Drs. Jonathan Katz and Bo Wang) were further backcrossed to NOD mice until 12 generations at the Scripps Research Institute. Mice of N10 to N12 backcross generation mice were intercrossed and were used in the experiments described herein. The heterozygous mice were intercrossed to generate homozygous (⫺/⫺), heterozygous (⫹/⫺), and wild-type (⫹/⫹) mice. The primers that were used to type mice for IL-10 gene disruption are as follows (www.jax.org): backward (oIMR086), 5⬘-GTG GGT GCA GTT ATT GTC TTC CCG-3⬘ (1723–1700 in exon I); forward (oIMR087), 5⬘-GCC TTC AGT ATA AAA GGG GGA CC-3⬘ (1523–1546) (in intron); and backward (oIMR088), 5⬘-CCT GCG TGC AAT CCA TCT TG-3⬘ (neocasette). A 200-bp PCR product indicates the wildtype allele and a 400-bp product indicates the mutant allele. Assessment of Diabetes Starting at 8 weeks of age, mice were tested for diabetes at 1-week or 2-week intervals by measuring blood glucose levels using a one-step Bayer Glucometer Elite (Bayer Corp., Elkhart, IN). Animals were considered diabetic when BG levels were ⬎300 mg/dl. Histological Analysis Lymphocytic infiltration of the islets was evaluated on hematoxylin and eosin (H&E)-stained paraffin sections of pancreas, taken at several levels throughout the organ. Paraffin-embedded sections of pancreata were stained with an immunoperoxidase method using polyclonal Abs to porcine insulin (Dako Corp., Carpinteria, CA), followed by a biotinylated secondary Ab and an avidin– biotin complex (Vector Laboratories, Inc., Burlingame, CA) as described earlier (7). Lymphocyte Proliferation Assays Splenocytes from indicated mice were cultured at 5 ⫻ 10 5 cells/well in 200 l of serum-free HL-1 medium (BioWhittaker, Walkersville, MD) supplemented with 100 units/ml penicillin, 100 g/ml streptomycin, 2 ⫻ 10 ⫺3 M L-glutamine, and 3 ⫻ 10 ⫺5 M 2-ME. The cells were cultured in 96-well flat-bottom microculture plates (Becton Dickinson, Franklin Lakes, NJ) in the presence of indicated Ags for 5 days (9). The cultures were pulsed with 1 Ci of [ 3H]TdR/well during the last 18 h of the assay and were later harvested. [ 3H]TdR uptake was measured in a  scintillation counter. The results were expressed as a stimulation index (SI), i.e., (mean cpm with Ag)/(mean cpm without Ag). OVA (Sigma Chemical Co., St. Louis, MO) was used as a control Ag. SI values ⬎3 over the background values were considered significant. Background values correspond to cpm obtained with the splenocyte cultures
FIG. 1. Genotyping of IL-10-deficient (⫺/⫺) and -sufficient (⫹/⫺, ⫹/⫹) NOD mice for IL-10 deficiency is done by PCR.
without GAD65. Recombinant GAD65 was prepared as described earlier (9). Cyclophosphamide (CYP) Injection CYP (Sigma) was dissolved in 0.9% NaCl at 20 mg/ml (on ice) immediately before injection. Eightweek-old IL-10-deficient (⫺/⫺) or -sufficient (⫹/⫹) NOD mice were injected intraperitoneally with cyclophosphamide at 200 mg/kg body wt (refers to 100 l of 20 mg/ml solution to 10 gm body wt) on days 0 and 14 (10). All mice were nondiabetic before CYP treatment. The treated mice were bled at weekly intervals for diabetes. Mice were considered diabetic if the BG levels were ⬎300 mg/dl. RESULTS IL-10-Deficient NOD Mice Readily Develop Insulitis Previous work from our labortatory has shown that neutralization of IL-10 with mAbs effectively prevented insulitis (4). To further establish the role of IL-10 in the development of insulitis in NOD mice, we generated IL-10-deficient NOD mice by backcrossing IL-10-deficient C57BL/6 mice with NOD mice for 10 generations. The genotyping of KO mice was verified by PCR and the results are shown in Fig. 1. IL-10deficient and -sufficient NOD mice were sacrificed at 12 weeks of age and their pancreata were embedded in 10% buffered formalin. As shown in Fig. 2, the islets from pancreata from IL-10- deficient NOD mice readily developed insulitis similar to that of their IL-10-sufficient (heterozygous) littermate controls. Additionally,
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TABLE 1 Histological Analysis of Pancreatic Sections from IL-10Deficient (ⴚ/ⴚ) and -Sufficient (ⴙ/ⴚ) NOD Mice Histological analysis of pancreatic islets Group IL-10-deficient (⫺/⫺) NOD IL-10-sufficient (⫹/⫺) NOD
Total
Normal
Peri-insulitis
Insulitis
90
30 (33%)
32 (36%)
28 (31%)
110
34 (31%)
36 (33%)
40 (36%)
Note. Indicated groups of female mice (n ⫽ 4) were sacrificed at 12 weeks of age. Their pancreata were paraffin-embedded and the sections were stained by H&E. Approximately 20 to 30 islets were scored for each mouse.
pothesized that IL-10 plays a role in the induction of T cell responses to islet Ags such as GAD65. To explore the role of IL-10 in the induction of T cell responses to graded doses of islet Ag GAD65 (from 40 g/ml to 2.5 g/ml), we performed T cell proliferation assays on IL-10 deficient (⫺/⫺) NOD mice. As shown in Fig. 3, splenocytes from IL-10-deficient (⫺/⫺) NOD mice readily responded to GAD65. The intensities of the proliferative responses to GAD65 between the groups were found to be comparable (P ⬎ 0.05). These findings show that IL-10’s role in the induction of T cell responses to GAD65 is dispensable. IL-10 Deficiency Does Not Accelerate Autoimmune Diabetes Previous findings showed that IL-4 deficiency failed to accelerate autoimmune diabetes in NOD mice (11). Similarly, we studied whether IL-10 deficiency would accelerate diabetes in IL-10-deficient NOD mice. If the
FIG. 2. Hematoxylin and eosin staining of formalin-fixed paraffin-embedded sections of pancreata from 12-week-old IL-10-sufficient (⫹/⫺) (A) and IL-10-deficient (⫺/⫺) (B) NOD mice. Note the development of insulitis in the pancreata from both groups of mice.
comparative insulitis scores of the pancreata between the groups are shown in the Table 1. There were no significant differences in the insulitis indexes between the groups. These findings demonstrate that, in contrast to expectations, IL-10 is not essential for the development of insulitis in NOD mice. T Cell Responses to Islet Antigen GAD65 in IL-10Deficient NOD Mice Are Intact Based on the finding that neutralization of IL-10 with mAbs effectively prevented insulitis (4), we hy-
FIG. 3. Proliferative responses of splenocytes against graded doses of GAD65 in vitro. Splenocytes from 8-week-old female IL-10sufficient (⫹/⫺) and IL-10-deficient (⫺/⫺) NOD mice were cultured in vitro against GAD65 for 5 days. The data represent means ⫾ SD of four mice/group. The lymphocyte proliferative responses observed against ovalbumin (control) did not differ from those results observed with splenocytes in the absence of Ag (data not shown).
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TABLE 2 IL-10 Deficiency Fails to Accelerate Spontaneous Autoimmune Diabetes in NOD Mice Incidence of diabetes (weeks) NOD mice
8
10
12
14
16
IL-10-deficient (⫺/⫺) IL-10 wild-type (⫹/⫹) IL-10 heterozygous (⫹/⫺)
0/9 nd nd
0/9 nd nd
0/9 nd nd
0/9 nd nd
0/9 1/10 1/10
18
20
24
Bleeding was stopped 2/10 2/10 6/10 1/10 3/10 5/10
28
32
% Incidence
7/10 8/10
7/10 8/10
0% 70% 80%
Note. Indicated groups of female NOD mice were measured for their blood glucose levels. Mice were considered diabetic if the BG levels were ⬎300 mg/dl. Note that IL-10-deficient (⫺/⫺) NOD mice were bled until 16 weeks of age; after that bleeding was discontinued because the majority of these mice were susceptibility to inflammatory bowel disease (as evidenced by loss of weight and rectal prolapse).
disease is accelerated, we should observe the onset of diabetes between 6 and 10 weeks of age. As shown in Table 2, over a 16-week period, none of the IL-10deficient (⫺/⫺) NOD mice showed accelerated diabetes (n ⫽ 9). During the same period of time, the wild-type (n ⫽ 10) and heterozygous (n ⫽ 10) littermate controls also failed to develop diabetes. However, the wild-type and heterozygous littermate controls progressed to diabetes over a 32-week period (70 to 80% incidence of diabetes). We could not monitor the IL-10-deficient NOD mice beyond a 16-week period for diabetes owing to their propensity to develop inflammatory bowel disease (evidenced by rectal prolapse) and continued body weight loss at as early as 10-weeks of age. Neverthless, the results show that IL-10 deficiency does not accelerate spontaneous diabetes in NOD mice. Cyclophosphamide (CYP) Provokes Accelerated Diabetes in IL-10-Deficient NOD Mice Since we could not monitor IL-10-deficient NOD mice for diabetes beyond the 16-week period owing to their early susceptibility to inflammatory bowel disease and lowered survival rate, we studied the role of IL-10 on cyclophosphamide-induced diabetes. Two doses of cyclophosphamide injected on days 0 and 14 are known to induce or accelerate diabetes in NOD mice (10). The IL-10-deficient (⫺/⫺) (n ⫽ 9) and -sufficient (wild-type ⫹/⫹) (n ⫽ 10) NOD mice were injected at 8 to 10 weeks of age with two doses of CYP at days 0 and 14. The injected mice were monitored for auto-
immune diabetes by measuring blood glucose levels. As shown in Table 3, CYP injection readily evoked diabetes in IL-10-deficient NOD mice. All the CYP-injected IL-10-deficient NOD mice developed diabetes at 1 week postinjection. In contrast, IL-10-sufficient NOD mice did not develop diabetes until 2 to 4 weeks postinjection (P ⬍ 0.05). Most of these mice in the latter group developed diabetes following the second dose of CYP. During the same period of time, PBS-treated control group IL-10-deficient (⫺/⫺) (n ⫽ 8) and IL-10-sufficient (⫹/⫹) (n ⫽ 8) NOD mice did not develop diabetes. These data demonstrate that deficiency of IL-10 accelerates CYP-induced diabetes. DISCUSSION The data presented here demonstrate that IL-10 is dispensable for induction of T cell responses to islet Ag GAD65 and autoimmune insulitis. IL-10 deficiency fails to accelerate diabetes. In contrast, IL-10 deficiency accelerates diabetes in NOD mice following cyclophosphamide injection. Our findings that IL-10 deficiency fails to accelerate spontaneous diabetes in NOD mice are in agreement with the results observed in IL-4-deficient NOD mice (11). The reason for the failure to observe accelerated diabetes in IL-10-deficient or IL-4-deficient NOD mice may be related to the compensation of other Th2 cytokines, e.g., the presence of IL-4 and IL-13 in IL-10deficient NOD mice and vice versa. The availability of
TABLE 3 IL-10 Deficiency Accelerates Cyclophosphamide-Induced Autoimmune Diabetes Incidence of diabetes (weeks) NOD mice
0
1
2
3
4
5
6
7
8
IL-10-deficient (⫺/⫺) IL-10-sufficient (⫹/⫹)
0/9 0/10
6/7 0/10
6/7 2/10
6/7 5/10
6/7 8/10
6/7 8/10
6/7 8/10
nd 8/10
nd 9/10
% Incidence 86% 90%
Note. Indicated groups of mice (8 weeks of age) of both sexes of N11 to N12 backcross generations were injected with cyclophosphamide at 20 mg/kg body wt on days 0 and 14. Blood glucose levels were measured weekly after the first injection. Mice were considered diabetic if the BG levels were ⬎300 mg/dl.
IL-10 DEFICIENCY, INSULITIS, AND DIABETES IN NOD MICE
NOD mice doubly mutant for IL-4 and IL-10 or the ability to block Th2 development by disrupting the Th2 signaling pathway via a transgenic approach would resolve whether Th2 cytokine deficiency accelerates diabetes in NOD mice. Alternatively, the susceptibility of IL-10-deficient NOD mice to inflammatory bowel disease might have obscured the acceleration of spontaneous diabetes in IL-10-deficient NOD mice. However, we feel that this latter issue may not be the case because IL-10-deficient (⫺/⫺) NOD mice develop insulitis similar to that of their littermate controls. Additionally, the splenocytes from IL-10-deficient (⫺/⫺) NOD mice have diabetogenic potential because these splenocytes could effectvely transfer diabetes into transgenic IL-10 –NOD-scid/scid mice. However, these splenocytes, upon injection into NOD-scid/scid mice, caused wasting disease and inflammatory bowel disease over a period of time (Balasa and Sarvetnick, unpublished data). Although IL-10 deficiency failed to accelerate spontaneous diabetes in NOD mice, CYP injection accelerated diabetes in IL-10-deficient NOD mice. The differences in these outcomes may relate to the differential thresholds of Th1 cytokines produced in non-CYP-injected IL-10-deficient NOD mice versus CYP-injected IL-10-deficient NOD mice. The observed accelerated diabetes in CYP-injected IL-10-deficient NOD mice may be related to the enhanced production of Th1 cytokines in vivo. This suggestion is in agreement with earlier findings that acceleration of autoimmune diabetes by CYP is associated with enhanced IFN-␥ secretion (12). Additionally, levels of Th1-promoting cytokines such as IL-18 (IFN-␥-inducing factor) and of IL12p40 mRNA have been detected in the adherent spleen cell population of cyclophosphamide-treated NOD mice (13, 14). As shown here, in some circumstances, IL-10 deficiency can indeed accelerate diabetes. The acceleration of diabetes in CYP-injected IL-10deficient NOD mice may relate to the loss of IL-10’s immunoregulatory or suppressive functions in vivo, such as suppression of macrophage costimulatory function and down-regulation of Th1-type cytokine production (15). In contrast to earlier studies in which anti-IL-10 Ab treatment effectively blocked insulitis in NOD mice (4), the current findings demonstrate that IL-10 deficiency did not prevent insulitis of NOD mice. Although this latter finding was unexpected, disparate outcomes between the two studies may relate to the compensatory mechanisms developed in IL-10-deficient NOD mice. Consistent with these observations, it was shown previously that neutralization of IFN-␥ blocked adoptively transferred diabetes (16), whereas IFN-␥ deficiency did not prevent autoimmune diabetes (17). In autoimmune diabetes of the NOD mouse, IL-10 has exhibited paradoxical effects. Neonatal expression of IL-10 in the islets accelerated diabetes begininng at
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4 to 5 weeks of age (5–7) and required autoreactive lymphocytes, as IL-10-NOD-scid/scid mice are resistant to autoimmune diabetes (7). Importantly, when autoreactive lymphocytes from NOD mice encountered IL-10, upon adoptive transfer, in the islets of transgenic IL-10 –NOD-scid/scid mice, the recipient mice readily developed diabetes (Balasa and Sarvetnick, unpublished observations). The proinflammatory role of IL-10 in autoimmune diabetes has been confirmed with IL-10-producing BDC2.5 Th2 cells (18), and double tg mice expressing BDC2.5 TCR and pancreatic IL-10 developed diabetes (19). However, other studies have documented immunosuppressive effects of IL-10 on autoimmune diabetes of NOD mice. A noncytolytic IL-10 fusion protein, upon intraperitoneal injection into young NOD mice, prevented their insulitis and diabetes owing to the suppression of Th1 cell responses (20). IL-10-transduced islet-specific Th1 cells could also prevent IDDM transfer in NOD mice (21). Systemic delivery of IL-10 by intramuscular injection of expression plasmid DNA prevented diabetes in NOD mice (22). Based on the available evidence, it can easily be said that timing of expression, location of expression, and concentration of IL-10 determines whether effects are immunostimulatory or immunosuppressive. In conclusion, our findings show that IL-10 is not absolutely required for insulitis and that its absence fails to accelerate spontaneous diabetes. However, its absence accelerates CYP-induced diabetes. ACKNOWLEDGMENTS We thank Drs. Ralph Kuhn and Werner Muller for providing IL-10-deficient mice on H-2 b background. We also thank Drs. Antonio Lacava and Christophe Benoist for critical comments on the manuscript.
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IL-10 Deficiency Does Not Inhibit Insulitis and Accelerates Cyclophosphamide-Induced Diabetes in the Nonobese Diabetic Mouse Balaji Balasa,* ,1 Kurt Van Gunst,* Nadja Jung,* Jonathan D. Katz,† and Nora Sarvetnick* *Department of Immunology, The Scripps Research Institute, La Jolla, California 92037; and †Department of Pathology, Center for Immunology, Washington University School of Medicine, St. Louis, Missouri 63110 Received February 24, 2000; accepted April 20, 2000
Cytokines produced by T cells and antigen-presenting cells (APC) modulate the immunopathogenesis of autoimmune diabetes. Understanding the role of cytokines will provide insights into the development of immunotherapeutic reagents for intervention in autoimmune diabetes. We and others have been focusing research on understanding the role of cytokines, in particular Th2 cytokine IL-10, in autoimmune diabetes. Work from our laboratory has shown that IL-10 is required for the development of diabetes because neutralization of IL-10 with antibodies effectively blocked insulitis development (4). Transgenic expression of IL-10 in the pancreatic islets of NOD mice accelerated their diabetes (5–7). These findings imply that IL-10 is actually an immunostimulatory factor in IDDM of the NOD mouse. To further explore the role of IL-10 in autoimmune diabetes, we introduced IL-10 gene disruption in NOD mice. We found that IL-10 deficiency does not inhibit insulitis. Splenocytes from IL-10-deficient NOD mice readily proliferated against glutamic acid decarboxylase 65 (GAD65). Importantly, IL-10-deficient NOD mice developed accelerated diabetes following cyclophosphamide (CYP) injection. These results demonstrate that IL-10 is not required for autoimmune diabetes.
IL-10 exterts profound immunostimulatory and immunoinhibitory effects. To explore the role of IL-10 in autoimmune diabetes of nonobese diabetic (NOD) mice, we generated IL-10-deficient NOD mice. In contrast to our previous results with neutralizing antibodies to IL-10, IL-10-deficient NOD mice developed insulitis and their splenocytes readily responded to islet antigen glutamic acid decarboxylase 65. IL-10deficient NOD mice did not develop accelerated spontaneous diabetes. On the other hand, IL-10-deficient NOD mice developed accelerated disease following cyclophosphamide (CYP) injection. These findings demonstrate that IL-10 is dispensable for autoimmune diabetes. IL-10’s absence fails to accelerate endogenous diabetes but potentiates CYP-induced diabetes. © 2000 Academic Press
Key Words: IL-10; insulitis; diabetes; NOD.
INTRODUCTION Insulin-dependent diabetes mellitus (IDDM) 2 is an autoimmune disease characterized by the loss of insulin-producing pancreatic  cells (1). The nonobese diabetic (NOD) mouse has been used as an animal model for IDDM in human beings (1). The autoimmune diabetes of the NOD mouse is a T-cell-mediated disease (2, 3). In its early and clinically silent phase, T cells and other immune cells infiltrate the islets, causing a progressive loss of  cells. When a majority of  cells have disappeared, the lack of insulin secretion leads to a failure of blood glucose (BG) homeostasis and diabetes.
MATERIALS AND METHODS Mice NOD/shi mice, 4 to 5 weeks old or 10 to 11 weeks old, were purchased from the rodent breeding colony at The Scripps Research Institute (La Jolla, CA). NOD-scid/ scid mice were also purchased from the rodent-breeding colony at The Scripps Research Institute.
1
B.B. is supported by postdoctoral fellowships from the Juvenile Diabetes Foundation International (JDFI) and the Myasthenia Gravis Foundation of America, Inc. This work was supported by a Diabetes Interdisciplinary Research Program grant from the JDFI (to N.S.). This is manuscript no. 12992-IMM. 2 Abbreviations used: IL-10, interleukin-10; BG, blood glucose; CYP, cyclophosphamide; GAD65, glutamic acid decarboxylase 65; IDDM, insulin-dependent diabetes mellitus; KO, knockout; NOD, nonobese diabetic; Ab, antibody.
Generation of IL-10-Deficient (⫺/⫺) Mice IL-10-deficient (⫺/⫺) C57BL/6 mice (8) (kindly provided by Dr. Ralph Kuhn and Werner Muller, Institute for Genetics, University of Cologne, Federal Republic of Germany) that were backcrossed to NOD mice for 9 97
0008-8749/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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generations (by Drs. Jonathan Katz and Bo Wang) were further backcrossed to NOD mice until 12 generations at the Scripps Research Institute. Mice of N10 to N12 backcross generation mice were intercrossed and were used in the experiments described herein. The heterozygous mice were intercrossed to generate homozygous (⫺/⫺), heterozygous (⫹/⫺), and wild-type (⫹/⫹) mice. The primers that were used to type mice for IL-10 gene disruption are as follows (www.jax.org): backward (oIMR086), 5⬘-GTG GGT GCA GTT ATT GTC TTC CCG-3⬘ (1723–1700 in exon I); forward (oIMR087), 5⬘-GCC TTC AGT ATA AAA GGG GGA CC-3⬘ (1523–1546) (in intron); and backward (oIMR088), 5⬘-CCT GCG TGC AAT CCA TCT TG-3⬘ (neocasette). A 200-bp PCR product indicates the wildtype allele and a 400-bp product indicates the mutant allele. Assessment of Diabetes Starting at 8 weeks of age, mice were tested for diabetes at 1-week or 2-week intervals by measuring blood glucose levels using a one-step Bayer Glucometer Elite (Bayer Corp., Elkhart, IN). Animals were considered diabetic when BG levels were ⬎300 mg/dl. Histological Analysis Lymphocytic infiltration of the islets was evaluated on hematoxylin and eosin (H&E)-stained paraffin sections of pancreas, taken at several levels throughout the organ. Paraffin-embedded sections of pancreata were stained with an immunoperoxidase method using polyclonal Abs to porcine insulin (Dako Corp., Carpinteria, CA), followed by a biotinylated secondary Ab and an avidin– biotin complex (Vector Laboratories, Inc., Burlingame, CA) as described earlier (7). Lymphocyte Proliferation Assays Splenocytes from indicated mice were cultured at 5 ⫻ 10 5 cells/well in 200 l of serum-free HL-1 medium (BioWhittaker, Walkersville, MD) supplemented with 100 units/ml penicillin, 100 g/ml streptomycin, 2 ⫻ 10 ⫺3 M L-glutamine, and 3 ⫻ 10 ⫺5 M 2-ME. The cells were cultured in 96-well flat-bottom microculture plates (Becton Dickinson, Franklin Lakes, NJ) in the presence of indicated Ags for 5 days (9). The cultures were pulsed with 1 Ci of [ 3H]TdR/well during the last 18 h of the assay and were later harvested. [ 3H]TdR uptake was measured in a  scintillation counter. The results were expressed as a stimulation index (SI), i.e., (mean cpm with Ag)/(mean cpm without Ag). OVA (Sigma Chemical Co., St. Louis, MO) was used as a control Ag. SI values ⬎3 over the background values were considered significant. Background values correspond to cpm obtained with the splenocyte cultures
FIG. 1. Genotyping of IL-10-deficient (⫺/⫺) and -sufficient (⫹/⫺, ⫹/⫹) NOD mice for IL-10 deficiency is done by PCR.
without GAD65. Recombinant GAD65 was prepared as described earlier (9). Cyclophosphamide (CYP) Injection CYP (Sigma) was dissolved in 0.9% NaCl at 20 mg/ml (on ice) immediately before injection. Eightweek-old IL-10-deficient (⫺/⫺) or -sufficient (⫹/⫹) NOD mice were injected intraperitoneally with cyclophosphamide at 200 mg/kg body wt (refers to 100 l of 20 mg/ml solution to 10 gm body wt) on days 0 and 14 (10). All mice were nondiabetic before CYP treatment. The treated mice were bled at weekly intervals for diabetes. Mice were considered diabetic if the BG levels were ⬎300 mg/dl. RESULTS IL-10-Deficient NOD Mice Readily Develop Insulitis Previous work from our labortatory has shown that neutralization of IL-10 with mAbs effectively prevented insulitis (4). To further establish the role of IL-10 in the development of insulitis in NOD mice, we generated IL-10-deficient NOD mice by backcrossing IL-10-deficient C57BL/6 mice with NOD mice for 10 generations. The genotyping of KO mice was verified by PCR and the results are shown in Fig. 1. IL-10deficient and -sufficient NOD mice were sacrificed at 12 weeks of age and their pancreata were embedded in 10% buffered formalin. As shown in Fig. 2, the islets from pancreata from IL-10- deficient NOD mice readily developed insulitis similar to that of their IL-10-sufficient (heterozygous) littermate controls. Additionally,
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TABLE 1 Histological Analysis of Pancreatic Sections from IL-10Deficient (ⴚ/ⴚ) and -Sufficient (ⴙ/ⴚ) NOD Mice Histological analysis of pancreatic islets Group IL-10-deficient (⫺/⫺) NOD IL-10-sufficient (⫹/⫺) NOD
Total
Normal
Peri-insulitis
Insulitis
90
30 (33%)
32 (36%)
28 (31%)
110
34 (31%)
36 (33%)
40 (36%)
Note. Indicated groups of female mice (n ⫽ 4) were sacrificed at 12 weeks of age. Their pancreata were paraffin-embedded and the sections were stained by H&E. Approximately 20 to 30 islets were scored for each mouse.
pothesized that IL-10 plays a role in the induction of T cell responses to islet Ags such as GAD65. To explore the role of IL-10 in the induction of T cell responses to graded doses of islet Ag GAD65 (from 40 g/ml to 2.5 g/ml), we performed T cell proliferation assays on IL-10 deficient (⫺/⫺) NOD mice. As shown in Fig. 3, splenocytes from IL-10-deficient (⫺/⫺) NOD mice readily responded to GAD65. The intensities of the proliferative responses to GAD65 between the groups were found to be comparable (P ⬎ 0.05). These findings show that IL-10’s role in the induction of T cell responses to GAD65 is dispensable. IL-10 Deficiency Does Not Accelerate Autoimmune Diabetes Previous findings showed that IL-4 deficiency failed to accelerate autoimmune diabetes in NOD mice (11). Similarly, we studied whether IL-10 deficiency would accelerate diabetes in IL-10-deficient NOD mice. If the
FIG. 2. Hematoxylin and eosin staining of formalin-fixed paraffin-embedded sections of pancreata from 12-week-old IL-10-sufficient (⫹/⫺) (A) and IL-10-deficient (⫺/⫺) (B) NOD mice. Note the development of insulitis in the pancreata from both groups of mice.
comparative insulitis scores of the pancreata between the groups are shown in the Table 1. There were no significant differences in the insulitis indexes between the groups. These findings demonstrate that, in contrast to expectations, IL-10 is not essential for the development of insulitis in NOD mice. T Cell Responses to Islet Antigen GAD65 in IL-10Deficient NOD Mice Are Intact Based on the finding that neutralization of IL-10 with mAbs effectively prevented insulitis (4), we hy-
FIG. 3. Proliferative responses of splenocytes against graded doses of GAD65 in vitro. Splenocytes from 8-week-old female IL-10sufficient (⫹/⫺) and IL-10-deficient (⫺/⫺) NOD mice were cultured in vitro against GAD65 for 5 days. The data represent means ⫾ SD of four mice/group. The lymphocyte proliferative responses observed against ovalbumin (control) did not differ from those results observed with splenocytes in the absence of Ag (data not shown).
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TABLE 2 IL-10 Deficiency Fails to Accelerate Spontaneous Autoimmune Diabetes in NOD Mice Incidence of diabetes (weeks) NOD mice
8
10
12
14
16
IL-10-deficient (⫺/⫺) IL-10 wild-type (⫹/⫹) IL-10 heterozygous (⫹/⫺)
0/9 nd nd
0/9 nd nd
0/9 nd nd
0/9 nd nd
0/9 1/10 1/10
18
20
24
Bleeding was stopped 2/10 2/10 6/10 1/10 3/10 5/10
28
32
% Incidence
7/10 8/10
7/10 8/10
0% 70% 80%
Note. Indicated groups of female NOD mice were measured for their blood glucose levels. Mice were considered diabetic if the BG levels were ⬎300 mg/dl. Note that IL-10-deficient (⫺/⫺) NOD mice were bled until 16 weeks of age; after that bleeding was discontinued because the majority of these mice were susceptibility to inflammatory bowel disease (as evidenced by loss of weight and rectal prolapse).
disease is accelerated, we should observe the onset of diabetes between 6 and 10 weeks of age. As shown in Table 2, over a 16-week period, none of the IL-10deficient (⫺/⫺) NOD mice showed accelerated diabetes (n ⫽ 9). During the same period of time, the wild-type (n ⫽ 10) and heterozygous (n ⫽ 10) littermate controls also failed to develop diabetes. However, the wild-type and heterozygous littermate controls progressed to diabetes over a 32-week period (70 to 80% incidence of diabetes). We could not monitor the IL-10-deficient NOD mice beyond a 16-week period for diabetes owing to their propensity to develop inflammatory bowel disease (evidenced by rectal prolapse) and continued body weight loss at as early as 10-weeks of age. Neverthless, the results show that IL-10 deficiency does not accelerate spontaneous diabetes in NOD mice. Cyclophosphamide (CYP) Provokes Accelerated Diabetes in IL-10-Deficient NOD Mice Since we could not monitor IL-10-deficient NOD mice for diabetes beyond the 16-week period owing to their early susceptibility to inflammatory bowel disease and lowered survival rate, we studied the role of IL-10 on cyclophosphamide-induced diabetes. Two doses of cyclophosphamide injected on days 0 and 14 are known to induce or accelerate diabetes in NOD mice (10). The IL-10-deficient (⫺/⫺) (n ⫽ 9) and -sufficient (wild-type ⫹/⫹) (n ⫽ 10) NOD mice were injected at 8 to 10 weeks of age with two doses of CYP at days 0 and 14. The injected mice were monitored for auto-
immune diabetes by measuring blood glucose levels. As shown in Table 3, CYP injection readily evoked diabetes in IL-10-deficient NOD mice. All the CYP-injected IL-10-deficient NOD mice developed diabetes at 1 week postinjection. In contrast, IL-10-sufficient NOD mice did not develop diabetes until 2 to 4 weeks postinjection (P ⬍ 0.05). Most of these mice in the latter group developed diabetes following the second dose of CYP. During the same period of time, PBS-treated control group IL-10-deficient (⫺/⫺) (n ⫽ 8) and IL-10-sufficient (⫹/⫹) (n ⫽ 8) NOD mice did not develop diabetes. These data demonstrate that deficiency of IL-10 accelerates CYP-induced diabetes. DISCUSSION The data presented here demonstrate that IL-10 is dispensable for induction of T cell responses to islet Ag GAD65 and autoimmune insulitis. IL-10 deficiency fails to accelerate diabetes. In contrast, IL-10 deficiency accelerates diabetes in NOD mice following cyclophosphamide injection. Our findings that IL-10 deficiency fails to accelerate spontaneous diabetes in NOD mice are in agreement with the results observed in IL-4-deficient NOD mice (11). The reason for the failure to observe accelerated diabetes in IL-10-deficient or IL-4-deficient NOD mice may be related to the compensation of other Th2 cytokines, e.g., the presence of IL-4 and IL-13 in IL-10deficient NOD mice and vice versa. The availability of
TABLE 3 IL-10 Deficiency Accelerates Cyclophosphamide-Induced Autoimmune Diabetes Incidence of diabetes (weeks) NOD mice
0
1
2
3
4
5
6
7
8
IL-10-deficient (⫺/⫺) IL-10-sufficient (⫹/⫹)
0/9 0/10
6/7 0/10
6/7 2/10
6/7 5/10
6/7 8/10
6/7 8/10
6/7 8/10
nd 8/10
nd 9/10
% Incidence 86% 90%
Note. Indicated groups of mice (8 weeks of age) of both sexes of N11 to N12 backcross generations were injected with cyclophosphamide at 20 mg/kg body wt on days 0 and 14. Blood glucose levels were measured weekly after the first injection. Mice were considered diabetic if the BG levels were ⬎300 mg/dl.
IL-10 DEFICIENCY, INSULITIS, AND DIABETES IN NOD MICE
NOD mice doubly mutant for IL-4 and IL-10 or the ability to block Th2 development by disrupting the Th2 signaling pathway via a transgenic approach would resolve whether Th2 cytokine deficiency accelerates diabetes in NOD mice. Alternatively, the susceptibility of IL-10-deficient NOD mice to inflammatory bowel disease might have obscured the acceleration of spontaneous diabetes in IL-10-deficient NOD mice. However, we feel that this latter issue may not be the case because IL-10-deficient (⫺/⫺) NOD mice develop insulitis similar to that of their littermate controls. Additionally, the splenocytes from IL-10-deficient (⫺/⫺) NOD mice have diabetogenic potential because these splenocytes could effectvely transfer diabetes into transgenic IL-10 –NOD-scid/scid mice. However, these splenocytes, upon injection into NOD-scid/scid mice, caused wasting disease and inflammatory bowel disease over a period of time (Balasa and Sarvetnick, unpublished data). Although IL-10 deficiency failed to accelerate spontaneous diabetes in NOD mice, CYP injection accelerated diabetes in IL-10-deficient NOD mice. The differences in these outcomes may relate to the differential thresholds of Th1 cytokines produced in non-CYP-injected IL-10-deficient NOD mice versus CYP-injected IL-10-deficient NOD mice. The observed accelerated diabetes in CYP-injected IL-10-deficient NOD mice may be related to the enhanced production of Th1 cytokines in vivo. This suggestion is in agreement with earlier findings that acceleration of autoimmune diabetes by CYP is associated with enhanced IFN-␥ secretion (12). Additionally, levels of Th1-promoting cytokines such as IL-18 (IFN-␥-inducing factor) and of IL12p40 mRNA have been detected in the adherent spleen cell population of cyclophosphamide-treated NOD mice (13, 14). As shown here, in some circumstances, IL-10 deficiency can indeed accelerate diabetes. The acceleration of diabetes in CYP-injected IL-10deficient NOD mice may relate to the loss of IL-10’s immunoregulatory or suppressive functions in vivo, such as suppression of macrophage costimulatory function and down-regulation of Th1-type cytokine production (15). In contrast to earlier studies in which anti-IL-10 Ab treatment effectively blocked insulitis in NOD mice (4), the current findings demonstrate that IL-10 deficiency did not prevent insulitis of NOD mice. Although this latter finding was unexpected, disparate outcomes between the two studies may relate to the compensatory mechanisms developed in IL-10-deficient NOD mice. Consistent with these observations, it was shown previously that neutralization of IFN-␥ blocked adoptively transferred diabetes (16), whereas IFN-␥ deficiency did not prevent autoimmune diabetes (17). In autoimmune diabetes of the NOD mouse, IL-10 has exhibited paradoxical effects. Neonatal expression of IL-10 in the islets accelerated diabetes begininng at
101
4 to 5 weeks of age (5–7) and required autoreactive lymphocytes, as IL-10-NOD-scid/scid mice are resistant to autoimmune diabetes (7). Importantly, when autoreactive lymphocytes from NOD mice encountered IL-10, upon adoptive transfer, in the islets of transgenic IL-10 –NOD-scid/scid mice, the recipient mice readily developed diabetes (Balasa and Sarvetnick, unpublished observations). The proinflammatory role of IL-10 in autoimmune diabetes has been confirmed with IL-10-producing BDC2.5 Th2 cells (18), and double tg mice expressing BDC2.5 TCR and pancreatic IL-10 developed diabetes (19). However, other studies have documented immunosuppressive effects of IL-10 on autoimmune diabetes of NOD mice. A noncytolytic IL-10 fusion protein, upon intraperitoneal injection into young NOD mice, prevented their insulitis and diabetes owing to the suppression of Th1 cell responses (20). IL-10-transduced islet-specific Th1 cells could also prevent IDDM transfer in NOD mice (21). Systemic delivery of IL-10 by intramuscular injection of expression plasmid DNA prevented diabetes in NOD mice (22). Based on the available evidence, it can easily be said that timing of expression, location of expression, and concentration of IL-10 determines whether effects are immunostimulatory or immunosuppressive. In conclusion, our findings show that IL-10 is not absolutely required for insulitis and that its absence fails to accelerate spontaneous diabetes. However, its absence accelerates CYP-induced diabetes. ACKNOWLEDGMENTS We thank Drs. Ralph Kuhn and Werner Muller for providing IL-10-deficient mice on H-2 b background. We also thank Drs. Antonio Lacava and Christophe Benoist for critical comments on the manuscript.
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