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*Diabetes Type 1
The Journal of Immunology, 2005, 175: 8401-8408.
Copyright © 2005 by The American Association of Immunologists

CXCL10 DNA Vaccination Prevents Spontaneous Diabetes through Enhanced {beta} Cell Proliferation in NOD Mice1

Toshikatsu Shigihara*, Akira Shimada2,*, Yoichi Oikawa*, Hiroyuki Yoneyama{dagger}, Yasuhiko Kanazawa*, Yoshiaki Okubo*, Kouji Matsushima{dagger}, Eiji Yamato{ddagger}, Jun-ichi Miyazaki{ddagger}, Akira Kasuga§, Takao Saruta* and Shosaku Narumi{dagger}

* Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan; {dagger} Department of Molecular Preventive Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan; {ddagger} Division of Stem Cell Regulation Research, Area of Molecular Therapeutics, Course of Advanced Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan; and § Department of Internal Medicine, Tokyo Denryoku Hospital, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CXCL10, a chemokine for Th1 cells, is involved in the pathogenesis of various Th1-dominant autoimmune diseases. Type 1 diabetes is considered to be a Th1-dominant autoimmune disease, and a suppressive effect of CXCL10 neutralization on diabetes development has been reported in a cyclophosphamide-induced accelerated diabetes model through induction of {beta} cell proliferation. However, intervention in a diabetes model might bring about opposite effects, depending on the timing, amount, or method of treatment. In the present study, we examined the effect of CXCL10 neutralization in a "spontaneous diabetes" model of NOD mice, using CXCL10 DNA vaccination (pCAGGS-CXCL10). pCAGGS-CXCL10 treatment in young NOD mice induced the production of anti-CXCL10 Ab in vivo and suppressed the incidence of spontaneous diabetes, although this treatment did not inhibit insulitis or alter the immunological response. pCAGGS-CXCL10 treatment enhanced the proliferation of pancreatic {beta} cells, resulting in an increase of {beta} cell mass in this spontaneous diabetes model as well. Therefore, CXCL10 neutralization is suggested to be useful for maintaining {beta} cell mass at any stage of autoimmune diabetes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Type 1 diabetes is considered to be a T cell-mediated autoimmune disease characterized by the selective destruction of pancreatic {beta} cells, resulting in insulin deficiency and hyperglycemia (1). Studies in the NOD mouse, which is an excellent animal model of human type 1 diabetes (2, 3), have suggested that Th1 cells are crucial in the initiation and amplification of autoimmune injury of {beta} cells.

Chemokines are involved in the pathogenesis of autoimmune disease in view of the selective attraction of various subsets of lymphocytes (4, 5). Based on structural motifs near their N-terminal cysteine residue, chemokines are divided into four subfamilies, termed CXC, CX3C, C, and CC. The target cells of chemokines are conferred by their chemokine receptors, which are expressed differentially. Th1/Tc1 (T cytotoxic 1) cells predominantly express CCR5 and CXCR3 (6, 7), whereas Th2/Tc2 cells express CCR3, CCR4, and CCR8 (6, 7, 8). Three ligands for CXCR3, CXCL10/IFN-{gamma} inducible protein 10 kDa, CXCL9/monokine induced by IFN-{gamma}, and CXCL11/IFN-inducible T cell {alpha} chemoattractant, have been identified (9, 10, 11, 12, 13). Among these three chemokines, CXCL10 is associated with the pathogenesis of various Th1-dominant diseases (14) such as experimental autoimmune encephalomyelitis (15, 16, 17), rheumatoid arthritis (18, 19), and infectious diseases (12, 20).

Recent studies revealed that CXCL10 is involved in the pathogenesis of type 1 diabetes. In humans, we have reported an elevated serum CXCL10 level in type 1 diabetic patients (21), followed by high-risk subjects for type 1 diabetes (22) and latent autoimmune diabetes in adults patients (23). In animal models, we have reported that CXCL10 neutralization suppressed the occurrence of autoimmune diabetes accelerated by cyclophosphamide (CY)3 in NOD mice through enhanced {beta} cell proliferation without affecting insulitis (24), whereas other groups reported that blockade of the CXCL10-CXCR3 interaction suppressed the development of insulitis in a virus-induced diabetes model (25, 26). However, our previous study has some critical issues from the viewpoint of application in clinical practice. CY treatment induces rapid progression of the disease course in NOD mice. This "accelerated" model identifies the so-called "malignant phase" (27, 28, 29) in type 1 diabetes, which definitely results in overt diabetes. However, in the human situation, it is sometimes hard to distinguish the "malignant phase" and "benign phase" (27, 28, 29), which may not result in overt diabetes. To apply CXCL10 neutralization to human type 1 diabetes, we have to confirm whether such an intervention can be used safely at any stage of type 1 diabetes because some interventions might bring about opposite effects depending on the timing, amount or method of treatment (30). Therefore, we evaluated the effect of neutralization of CXCL10 in the earlier, benign phase of type 1 diabetes. In the present study, we used a system of gene transfer into muscle with electroporation (31) to induce the production of anti-chemokine Ab in vivo for a long period and evaluated the effect of treatment on spontaneous diabetes development in NOD mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Four-week-old female NOD mice were purchased from CLEA Japan. They were kept under specific pathogen-free conditions in the animal facility of Tokyo Denryoku Hospital. Urinary glucose analysis was performed weekly using Tes-tape (Shionogi). Blood glucose level was determined using Glutest-Ace (Sanwa Kagaku) when glycosuria was detected, and mice were considered to be diabetic after obtaining two consecutive, 1 wk apart, blood glucose values > 250 mg/dl. In our colony, the cumulative incidence of spontaneous diabetes development in female NOD mice reaches ~70% by 40 wk of age.

Plasmid vectors

Plasmid pCAGGS-CXCL10 was constructed by inserting the rat CXCL10 cDNA, in which one amino acid (92nd) of its deduced amino acid sequence was replaced with that of mouse CXCL10 (10), into the unique EcoRI site between the CAG (cytomegalovirus immediate-early enhancer-chicken {beta}-actin hybrid) promoter and the 3'-flanking sequence of the rabbit {beta}-globin gene of the pCAGGS expression vector (Fig. 1). By immunizing mice with this partially modified rat CXCL10, we obtained a mAb, {alpha}rIPb, which was confirmed to neutralize not only rat but also mouse CXCL10 (10, 24, 31). Plasmids were grown in E. coli HB101, prepared using Qiagen plasmid purification columns (Qiagen), according to the supplier’s protocol, and further purified by ethidium bromide CsCl equilibrium density gradient centrifugation. The quantity and quality of the purified plasmid DNA were assessed by the OD at 260 and 280 nm and also by electrophoresis in 1% agarose gel. The expression capacity of the resulting pCAGGS-CXCL10 plasmid DNA was confirmed by transient transfection into 293 cells, and the transcript was detected in 293 cells and in the culture medium by Western blotting using {alpha}rIPb (10) (data not shown).



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  		FIGURE 1. Structure of pCAGGS-CXCL10 plasmid DNA. pCAGGS-CXCL10 plasmid DNA was constructed by inserting the rat CXCL10 cDNA into the unique EcoRI site between the CAG promoter and the 3'-flanking sequence of the rabbit {gamma}-globin gene of the pCAGGS expression vector.

 
Intramuscular injection with electroporation

Four-week-old NOD mice were anesthetized with pentobarbital, and 50 µg of pCAGGS-CXCL10 or pCAGGS plasmid DNA (pCAGGS-control) was injected into the bilateral tibialis anterior muscles (total 100 µg of plasmid DNA/mouse) with the electroporation methods as described previously (30, 32). These treatments were repeated sequentially 2 wk later (at 6 wk of age).

Evaluation of anti-CXCL10 Ab in serum of pCAGGS-CXCL10-treated mice

A direct ELISA was used to determine the anti-rat CXCL10 Ab titer or anti-mouse CXCL10 Ab titer in pCAGGS-CXCL10 or pCAGGS-control-treated mice. Recombinant rat CXCL10 (10) or mouse CXCL10 (PeproTech) was used to coat a 96-well ELISA plate (Nunc) at a concentration of 50 ng/well. Diluted serum (1/64 or 1/8, respectively) from treated mice was added to the plate. Sheep anti-mouse Ig HRP-conjugated Ab (Amersham Biosciences) was used as a labeled Ab. Tetramethylbenzidine (BD Pharmingen) was used as a soluble HRP substrate.

Evaluation of insulitis

The pancreas was removed from each mouse, fixed in 10% formaldehyde, and embedded in paraffin. Thin sections at five levels, 150 µm apart, were cut for staining with H&E to evaluate the islet-infiltrating immune cells by light microscopy. At least 30 islets from each mouse were observed and scored by two independent blinded observers using the following criteria: grade 0, islet free of insulitis; grade 1, peri-insulitis; grade 2, intrainsulitis with mononuclear cell infiltration of <50%; and grade 3, intrainsulitis with mononuclear cell infiltration of ≥50% of the area of each islet.

Immunohistochemical staining

The pancreas from each nondiabetic NOD mouse was obtained at 16 wk of age when mice were killed for evaluation of several parameters. For in vivo proliferation assay, mice were i.p. injected with BrdU (Sigma-Aldrich) (2 mg/mouse) 1 h before killing. Each pancreas was inflated with OCT compound and snap-frozen in liquid nitrogen.

To identify the cell type of pancreatic islet-infiltrating cells, FITC-labeled anti-mouse CD4 mAb (clone H129.19; BD Pharmingen) and PE-labeled anti-mouse CD8 mAb (53-6.7; BD Pharmingen) were used. Four-percent paraformaldehyde-fixed frozen tissue sections were cut at 6 µm and incubated with each mAb. Then they were examined by fluorescence microscopy.

To detect replicating cells, a BrdU staining kit (Zymed Laboratories) was used, according to the manufacturer’s instructions (24). Before BrdU staining was performed, acetone-fixed 6-µm frozen tissue sections were immunostained with rabbit anti-insulin polyclonal Ab (Santa Cruz Biotechnology) using indirect immunoalkaline phosphatase methods. To further confirm whether BrdU+ cells were {beta} cells, double immunofluorescence analysis for BrdU and pancreatic duodenal homeobox (PDX-1) was performed. Four-percent paraformaldehyde-fixed 6-µm fresh frozen tissue sections were incubated with biotinylated BrdU Ab (Zymed Laboratories) and anti-PDX-1 polyclonal Ab (Trans Genic), followed by Alexa-488-labeled streptavidin and Alexa-568-labeled anti-rabbit IgG, respectively (Molecular Probes).

Furthermore, to quantitate insulin content, we calculated the ratio of insulin-positive area to the whole islet area using Scion Image (Scion Corporation).

Polyclonal stimulation of splenocytes

The spleen was removed aseptically from each mouse and minced. After lysing RBC, cells were resuspended in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FBS (Invitrogen Life Technologies) and penicillin/streptomycin (Invitrogen Life Technologies). A part of the splenocytes was used to evaluate the proportion by flow cytometry. Then, 1 x 106 cells resuspended in 200 µl of culture medium were transferred to each well of a round-bottom 96-well plate. Then, anti-CD3 Ab (145-2C11; BD Pharmingen) was added to each well (final concentration: 5 µg/ml). The cells were cultured for 48 h at 37°C in a humidified 5% CO2 atmosphere. The supernatant was collected at the end of culture and frozen at –80°C until cytokine assay.

Flow cytometry

FITC-labeled anti-mouse CD4 mAb (H129.19; BD Pharmingen), and PE-labeled anti-mouse CD8 mAb (53-6.7; BD Pharmingen), PE-labeled anti-mouse CD25 mAb (PC61; BD Pharmingen) were used for staining of lymphocytes. The prepared cells were analyzed with an Epics Altra (Corixa).

Cytokine measurement by ELISA

Each cytokine (IFN-{gamma}, IL-4, IL-10) was measured by ELISA as described previously (33). Briefly, a flat-bottom 96-well plate (Nunc) was coated with anti-IFN-{gamma}, anti-IL-4, or anti-IL-10 Abs (BD Pharmingen) with overnight incubation at 4°C. The collected supernatant and standards (purified rIFN-{gamma}, IL-4, IL-10; BD Pharmingen) were added, and the plate was incubated for another 2 h. After washing, biotinylated anti-IFN-{gamma}, anti-IL-4, or anti-IL-10 Abs (BD Pharmingen) were added, followed by another 1-h period of incubation. AB solution (Vecstatin ABC kit; Vector Laboratories) was then added, and the plate was incubated for 30 min. Finally, the color reagent (tetramethylbenzidine substrate; BD Pharmingen) was added, and the OD was measured with an ELISA reader (Bio-Rad) at 450 nm. The amount of cytokine present was determined from standard curves.

Semiquantitative RT-PCR

Total RNA was extracted from the pancreas, pancreatic lymph nodes (pLN) or spleen using an RNeasy Mini kit (Qiagen). During the procedure, DNase treatment was performed according to the manufacturer’s protocol. The extracted RNA was reverse transcribed using NotI-d(T)18 primer and a First-Strand cDNA synthesis kit (Amersham Biosciences), according to the manufacturer’s instructions. Semiquantitative RT-PCR was conducted for IFN-{gamma}, IL-4, IL-10, TNF-{alpha}, IL-1{beta}, TGF-{beta}, CXCR3, CXCL10, CXCL9, CXCL11, and GAPDH (internal control) in an ABI Prism 7700 sequence detector (PE Applied Biosystems, Japan). The primer and probe sequences used were as previously described (24, 33). All reactions were performed using a TaqMan Universal MasterMix (Applied Biosystems). The obtained mRNA level was expressed relative to that of the GAPDH PCR product amplified from the same sample ((sample PCR product/GAPDH PCR product) x constant).

Ag-specific stimulation and intracellular cytokine staining

Ag-specific cytokine responses were assessed using an intracellular cytokine staining system as described previously (30). Briefly, the spleen was removed aseptically and minced. After lysing RBC, cells were resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and penicillin/streptomycin. The cells were transferred to 5-ml polystyrene round-bottom tubes (BD Biosciences). Then, recombinant glutamic acid decarboxylase 65 (GAD 65) produced in yeast (RSR) was added to each tube (final concentration: 5 µg/ml). Endotoxin level of recombinant GAD 65 was <0.1 EU/ml (at Ag concentration of 10 µg/ml) by a chromogenic assay method. No stimulant was added to control tubes. Then, the cells were incubated at 37°C in a humidified 5% CO2 atmosphere for a total of 72 h, with the last 4 h, including a final concentration of 10 µg/ml brefeldin A (Sigma-Aldrich). After incubation, 5 µl of CD4-CyChrome Ab (H129.19; BD Pharmingen) was added, and the tubes were incubated at room temperature for 15 min. Then, 4 ml of FACS lysing solution (BD Biosciences) was added, and the tubes were vortexed gently and incubated at room temperature for 10 min. After centrifuging the tubes, the supernatant was removed, and the cells were washed with 0.1% BSA-PBS. Then, 500 µl of FACS permeabilizing solution (BD Biosciences) was added, and the tubes were incubated for 10 min at room temperature in the dark. After washing twice with 0.1% BSA-PBS, 3 µl of IFN-{gamma}-FITC Ab (IgG1; BD Pharmingen) or isotype control Abs (IgG1-FITC; BD Pharmingen) was added, and the tubes were incubated for 30 min at room temperature in the dark. After washing, the prepared cells were analyzed with an Epics Altra (Corixa).

Statistical analysis

Results are presented as mean ± SEM. Log-rank test was used to compare the incidence of diabetes. Other mean values in the pCAGGS-CXCL10 and pCAGGS plasmid DNA groups were compared by Mann-Whitney U test or, in some instances, by ANOVA. A value of p < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
pCAGGS-rat CXCL10 plasmid DNA treatment induces production of anti-mouse CXCL10 Ab in vivo

First, to determine whether pCAGGS-rat CXCL10 plasmid DNA (pCAGGS-CXCL10) or pCAGGS plasmid DNA (pCAGGS-control) treatment induces an immunological reaction in vivo, the serum level of anti-rat CXCL10 Ab in mice was examined after plasmid DNA injection (Fig. 2A). The anti-rat CXCL10 Ab level gradually increased only in mice treated with pCAGGS-CXCL10, and a high level of anti-rat CXCL10 Ab was maintained for more than several months after injections (p < 0.0001 vs pCAGGS-control). Then, we examined the anti-mouse CXCL10 Ab level to determine whether pCAGGS-CXCL10 treatment induces the production of Abs reacting to mouse CXCL10 as well. The anti-mouse CXCL10 Ab level in mice treated with pCAGGS-CXCL10 was significantly higher than that in mice treated with pCAGGS-control on days 28 and 56, respectively (OD 0.97 ± 0.23 vs 0.07 ± 0.00, p < 0.01 on day 28; 0.46 ± 0.15 vs 0.12 ± 0.01, p < 0.01 on day 56; Fig. 2B). Thus, we confirmed that pCAGGS-CXCL10 treatment induces the production of anti-mouse CXCL10 Ab in NOD mice.



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  		FIGURE 2. pCAGGS-CXCL10 plasmid DNA injection augments anti-CXCL10 Abs. pCAGGS-CXCL10 plasmid DNA was injected on days 0 and 14 (arrows), and serum samples were examined to confirm the development of Ab to CXCL10 at different time points. A, Change in rat CXCL10Ab titer in NOD mice treated with pCAGGS-CXCL10 or pCAGGS (control) plasmid DNA. Results are shown as mean ± SEM (n = 3/group). **, p < 0.0001 by repeated measures ANOVA. B, Mouse CXCL10 Ab titer in NOD mice treated with pCAGGS-CXCL10 or pCAGGS (control) plasmid DNA on days 28 and 56. Results are shown as mean ± SEM (n = 5/group). *, p < 0.01 by Mann-Whitney U test.

 
pCAGGS-rat CXCL10 plasmid DNA treatment suppresses incidence of spontaneous diabetes in NOD mice

To determine the effect of pCAGGS-CXCL10 treatment on diabetes incidence, pCAGGS-CXCL10 (CXCL10 group; n = 23) and pCAGGS-control (control group; n = 24) mice were injected at 4 and 6 wk of age. The earliest development of overt diabetes in the CXCL10 group was observed 7 wk later than that in the control group. The cumulative incidence of diabetes was significantly decreased in the CXCL10 group (26%, 6 of 23) as compared with that in the control group (58%, 14 of 24) at 30 wk of age (p < 0.02; Fig. 3).



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  		FIGURE 3. pCAGGS-CXCL10 plasmid DNA treatment suppresses incidence of spontaneous diabetes in NOD mice. Female NOD mice were injected sequentially with pCAGGS-control (control group; n = 24) or pCAGGS-CXCL10 (CXCL10 group; n = 23) in the bilateral tibialis anterior muscles (total 100 µg of plasmid DNA/mouse) with electroporation at 4 and 6 wk of age (arrows in figure). *, p < 0.02 vs control group at 30 wk of age by log-rank test.

 
pCAGGS-rat CXCL10 plasmid DNA treatment does not affect degree of insulitis

To investigate the mechanism of the suppressive effect of pCAGGS-CXCL10 injections on a spontaneous diabetes NOD mouse model, the degree of insulitis in the CXCL10 group as well as the control group was evaluated at 8, 12, and 16 wk of age. Contrary to our expectations, insulitis score in the CXCL10 group was not significantly different from that in the control group at each time point (Fig. 4, A and B). Therefore, to determine whether the phenotype of islet-infiltrating cells is different or not in these groups, immunohistochemical staining of the pancreas was performed at 16 wk of age. T cells infiltrating islets were predominantly CD4+ cells rather than CD8+ cells, and the degree of infiltration of CD8+ cells in islets was essentially the same in the CXCL10 and the control groups (Fig. 4C).



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  		FIGURE 4. Evaluation of degree of insulitis. A, The pancreas was obtained from the pCAGGS-control (control) and pCAGGS-CXCL10-injected (CXCL10) groups at 16 wk of age and stained with H&E (magnification, x100). B, Insulitis score in both groups was evaluated at 8, 12, and 16 wk of age (n = 5/group). All of these mice were nondiabetic. Grade of insulitis was scored according to the following criteria; 0, islet free of insulitis; 1, peri-insulitis; 2, intrainsulitis with mononuclear cell infiltration of <50%; and 3, intrainsulitis with mononuclear cell infiltration of ≥50% of the area of each islet. C, CD4-positive cells (green) and CD8-positive cells (red) infiltrating the pancreatic islets in both groups were evaluated at 16 wk of age (magnification, x200).

 
Furthermore, to compare cytokine expression levels in the pancreas, we examined the mRNA expression of IFN-{gamma}, IL-4, IL-10, TNF-{alpha}, IL-1{beta}, and TGF-{beta} in both groups at 16 wk of age (Fig. 5A). IFN-{gamma} mRNA expression in the pancreas in the CXCL10 group was rather higher than that in the control group, although the difference between the groups was not statistically significant. Other cytokine mRNA expression levels in the pancreas in the CXCL10 group were similar to those in the control group. We also examined the mRNA expression level of CXCR3 in the pancreas (Fig. 5A). CXCR3 mRNA expression level in the pancreas was similar in both groups. The expression levels of other CXCR3 ligands, CXCL9 and CXCL11, in the pancreas were also similar in both groups.



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  		FIGURE 5. Cytokines, chemokines, and CXCR3 mRNA expression levels in the pancreas (A) and the pLN (B). mRNA expression levels of each cytokine, chemokine, and CXCR3 in the pancreas (n = 10/group) (A) and pLN (B) from the pCAGGS-control group (control) and pCAGGS-CXCL10-injected group (CXCL10) were evaluated at 16 wk of age using semiquantitative PCR analysis. pLN from five mice per group were pooled and used for analysis. The mRNA level was expressed relative to the level of GAPDH PCR product amplified from the same sample. Data obtained from three experiments are shown as mean ± SEM.

 
pCAGGS-rat CXCL10 plasmid DNA does not affect status of pLN

To determine the status of draining lymph nodes, we investigated the mRNA expression levels in pLN in both groups at 16 wk of age. IFN-{gamma}, IL-4, IL-10, TNF-{alpha}, IL-1{beta}, TGF-{beta}, and CXCR3 mRNA expression levels in pLN in the CXCL10 group were not significantly different from those in the control group (Fig. 5B). CXCL9 and CXCL11 mRNA expression levels in pancreatic LN were similar in both groups.

Cytokine profiles upon polyclonal or Ag-specific stimulation

To examine the effect of pCAGGS-CXCL10 plasmid DNA on the systemic Th1/Th2 cytokine balance, we evaluated the cytokine production of splenocytes induced by polyclonal (anti-CD3 Ab) stimulation at 16 wk of age. As shown in Fig. 6A, IFN-{gamma} and IL-4 levels were not significantly different between the CXCL10 and control group at 16 wk of age. IL-10 level in the CXCL10 group was a little higher than that in the control group, although the difference was not statistically significant. There was no significant difference in the population of splenic CD4+ and CD8+ cells between the CXCL10 and control groups (data not shown). To determine whether regulatory T cells are involved in the suppressive effect of pCAGGS-CXCL10 treatment on diabetes development, we evaluated the population of CD4+CD25+ cells in the spleen and pancreatic lymph node by flow cytometry. The population of CD4+CD25+ cells in the spleen in the CXCL10 group was similar to that in the control group (7.35 ± 0.82% vs 6.46 ± 1.28% (per CD4+ cells)). In pLN also, the population of CD4+CD25+ cells in the CXCL10 group was similar to that in the control group (9.29 vs 9.38% (per CD4+ cells); Fig. 6B).



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  		FIGURE 6. Cytokine profiles of splenocytes upon anti-CD3 Ab stimulation (A) and population of CD4+CD25+ cells (B). A, Stimulation of splenocytes from the pCAGGS-control group (control) and pCAGGS-CXCL10-injected group (CXCL10) was performed at 16 wk of age (n = 5/group). Cytokines in the supernatants were measured by ELISA. Data are shown as mean ± SEM. B, The population of CD4+CD25+ cells in the spleen (n = 5/group) and pLN at 16 wk of age in both groups. pLN from five mice per group were pooled and used for analysis. Data are shown as mean ± SEM.

 
Next, to determine the effect of pCAGGS-CXCL10 treatment on the development of islet-associated Ag-reactive T cells, we evaluated the population of GAD-reactive IFN-{gamma}-producing CD4+ cells using an intracellular cytokine staining system. The number of GAD-reactive IFN-{gamma}-producing CD4+ cells in the spleen was lower in the CXCL10 group than that in the control group at 16 wk of age, but the difference was not statistically significant (4.50 ± 3.98 vs 10.00 ± 3.73/50,000 CD4+ cells).

pCAGGS-rat CXCL10 plasmid DNA treatment enhances proliferation of pancreatic {beta} cells in NOD mice

Because pCAGGS-CXCL10 treatment did not modulate immunological responses in NOD mice, we examined whether another mechanism could explain the suppressive effect of this treatment on diabetes development. We have observed previously the expression of CXCL10 in pancreatic {beta} cells and reported that CXCL10 neutralization enhanced the proliferative response of {beta} cells in a CY-induced NOD mouse model, a "malignant phase" model (24). Therefore, we hypothesized that pCAGGS-CXCL10 treatment in the earlier stages may have enhanced the proliferation of pancreatic {beta} cells in a spontaneous diabetes model, in a "benign phase" model, in NOD mice as well. We examined the effect of pCAGGS-CXCL10 injections on the proliferation of pancreatic {beta} cells in vivo by BrdU labeling 1 h before killing mice in both the CXCL10 group and the control group at 16 wk of age. BrdU+ islet cells were regarded as proliferative cells. BrdU+ cells were significantly increased in islets in the CXCL10 group compared with those in the control group (2.16 ± 0.25/mm2 (x10–2) vs 1.07 ± 0.36/mm2 (x10–2), p < 0.03; Fig. 7, A and C). BrdU+ cells were mainly found in islets with infiltration of mononuclear cells. Some BrdU+ cells in the islets were also stained with anti-insulin Ab, either strongly or faintly. The size of the nucleus of BrdU+ cells was mostly larger than that of lymphocytes. We speculated that BrdU+ cells with faint (or no) insulin staining were immature {beta} cells and/or islet cells, as we observed in a previous study (24). To confirm whether BrdU+ cells observed in islets were indeed {beta} cells, double staining of PDX-1 and BrdU was performed. We found that most BrdU+ cells were PDX-1 positive (Fig. 7B) and could thus confirm that most BrdU+ cells were {beta} cells.



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  		FIGURE 7. pCAGGS-CXCL10 plasmid DNA treatment enhances proliferation of pancreatic {beta} cells. A, Immunohistochemical evaluation of the pancreas in the pCAGGS-control group (control) and pCAGGS-CXCL10-injected group (CXCL10) at 16 wk of age. BrdU+ cells (brown) and insulin-positive cells (blue) are shown (magnification, x400). Arrows indicate BrdU+ cells. B, Fluorescent-immunohistochemical evaluation of the pancreas at 16 wk of age was performed. PDX-1+ cells (red) and BrdU+ cells (green) are shown (magnification, x200). Arrows indicate double-positive (PDX-1+BrdU+) cells. C, The number of BrdU+ cells in each islet was counted and divided by the area of the islet (n = 5/group). Values are shown as mean ± SEM. *, p < 0.05 (by Mann-Whitney U test). D, The ratio of insulin-positive area to the whole islet area was examined in both groups using Scion Image. More than 40 islets were examined in each group. Values are shown as mean ± SEM. **, p < 0.001 (by Mann-Whitney U test).

 
In contrast, to determine whether pCAGGS-CXCL10 treatment affects apoptosis of pancreatic {beta} cells, insulin staining and TUNEL were performed on the pancreas from both groups, and we found that TUNEL was negative in pancreatic {beta} cells in both groups.

Lastly, to quantitate the "{beta} cell mass," the ratio of insulin-positive area to the whole islet area was evaluated after immunohistochemical staining for insulin in both groups of 16-wk-old nondiabetic mice. The "{beta} cell mass" in the pancreas in the CXCL10 group was significantly larger than that in the control group (71.43 ± 4.73 vs 47.43 ± 4.22%, p < 0.001; Fig. 6D), corresponding to the number of BrdU+ replicating cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present study showed that CXCL10 DNA vaccination induces the production of anti-CXCL10 Ab in vivo and suppresses the onset of spontaneous diabetes through enhanced {beta} cell proliferation in NOD mice. These results suggest that CXCL10 neutralization in the early stage, in the "benign phase," as well as in a CY-induced diabetes model, in the "malignant phase" (24), is effective for prevention of spontaneous diabetes through the enhancement of {beta} cell replication in islets with inflammation.

A plasmid chemokine DNA injection system was used to reduce invasiveness and to maintain the effect of neutralization for a long period of time in the present study. A plasmid DNA injection system is used for delivery of gene products themselves or for eliciting an immune response against gene products depending on the nature of the selected plasmid (34). Although we have used pCAGGS-murine cytokine DNA treatment to deliver a cytokine itself systemically, the "immune response" against produced cytokines had never been observed in our previous studies (30, 35, 36). Therefore, we decided to use rat instead of murine CXCL10 cDNA, in the expectation of eliciting a vaccination-like effect because the overall identity of rat CXCL10 to murine CXCL10 is ~80% at the amino acid level, and we have already obtained a mouse CXCL10-neutralizing Ab by immunization of rat CXCL10 (10). In the present study, pCAGGS-rat CXCL10 plasmid DNA treatment induced significant Ab production against mouse CXCL10 as well as against rat CXCL10 in the treated NOD mice (Fig. 2), suggesting that the produced anti-mouse CXCL10 Ab could neutralize mouse CXCL10 in the treated NOD mice.

Pancreatic {beta} cells produce CXCL10 under stress such as exposure to proinflammatory cytokines and/or lymphocyte infiltration (24, 25). The pancreatic CXCL10 expression level was positively correlated with both the degree of insulitis and the pancreatic IFN-{gamma} expression level and was also positively correlated with the population of GAD-reactive IFN-{gamma}-producing CD4+ splenocytes in prediabetic NOD mice (30). However, CXCL10 neutralization did not affect the degree of insulitis or CXCR3 expression in the pancreas in spontaneous diabetes (Figs. 4 and 5A) as well as in CY-induced diabetes of NOD mice (24), although blockade of the CXCL10-CXCR3 interaction inhibited the infiltration of lymphocytes into islets and suppressed the onset of diabetes in receptor interacting protein-lymphocytic choriomeningitis virus transgenic mice (25, 26). Moreover, in pLN, which may be a major site of priming and activation of diabetogenic T cells (37, 38), pCAGGS-CXCL10 treatment did not alter mRNA expression levels of CXCR3 and proinflammatory cytokines (Fig. 5B). Furthermore, the numbers of GAD-reactive IFN-{gamma}-producing CD4+ cells and the population of CD4+CD25+ cells in pCAGGS-CXCL10-treated mice were not significantly different from those in pCAGGS-control-treated mice (Fig. 6, A and B). However, it is noteworthy that even though we could not find any difference regarding the population of CD4+CD25+ cells between the two groups, the population of CD4+CD25+ cells may not represent accurately altering the regulatory T cell pool in pCAGGS-CXCL10-treated mice; thus, more detailed examination such as FoxP3 staining may be necessary to conclude the alteration of the regulatory T cell pool by pCAGGS-CXCL10 treatment. Regarding progression of insulitis, not only T cells but also B cells, dendritic cells, macrophages, and/or NK cells play roles in NOD mice. Even though we could not find any immunological differences between the CXCL10 and the control group, it is impossible to completely rule out the involvement of these immune cells in the suppressive effect of CXCL10 vaccination. However, we have already demonstrated that CXCL10 neutralization did not alter the population of islet-infiltrating cells (CD4, CD8, macrophage, NK cell) in the insulitis region in our previous study (24); therefore, we speculate that CXCL10 vaccination did not alter the population of these immune cells in the present study as well. As compared with the receptor interacting protein-lymphocytic choriomeningitis virus transgenic mouse model, whether CXCR3+ effector cells play an essential role in the progression of insulitis may be dependent on whether the immunogen is an external or intrinsic self Ag and whether the occurrence of diabetes is acute or chronic (24). Furthermore, differences in the genetic background between the NOD mouse and C57BL/6 mouse might be an important factor. In the NOD mouse, replacement of an Idd gene, except the MHC region, does not suppress spontaneous insulitis completely; therefore, multiple background genes might be involved in the progression of insulitis in the NOD mouse (39, 40). Although differences in the dose, timing, or route of intervention can lead to different outcomes in the development of diabetes in NOD mice (30), our results at least suggest that pCAGGS-CXCL10 treatment did not aggravate the immunological response against {beta} cells in spontaneous diabetes as well as in a CY-induced diabetes model in NOD mice (15) and that "CXCL10 neutralization therapy" can be used to prevent autoimmune diabetes irrespective of the timing of intervention.

Recent studies suggest that CXCL10 possesses not only the effect of chemotaxis but also the effect of cell proliferation. Regarding cell proliferation, our previous studies indicated that CXCL10 neutralization induced a cellular proliferative response (24, 41), whereas some suggested that CXCL10 itself was associated with regeneration of tissues (42). Regarding the opposite effect of CXCL10 among the reports, it has been reported that an alternatively splicing variant of CXCR3 (renamed CXCR3-A), named CXCR3-B, was identified, and that CXCR3-A mediates the increase in survival of vascular endothelial cells, whereas CXCR3-B mediates the inhibition of cell growth induced by ligands of CXCR3 (42). Therefore, differences in the type of CXCR3 may explain the different effects of CXCL10, although more detailed studies are required to reach a conclusion. In the pancreas, we have reported that CXCL10 and CXCR3 are coexpressed in {beta} cells in infiltrated islets and that CXCL10 neutralization enhanced {beta} cell replication in islets in a CY-induced NOD model (24). In the present study also, CXCL10 neutralization by pCAGGS-CXCL10 plasmid DNA treatment enhanced islet cell proliferation and increased the {beta} cell mass (Fig. 7). We found that most BrdU+ cells in islets were PDX-1+ cells, and apoptotic cells were not detected in islets in either group, indicating that enhanced {beta} cell replication by pCAGGS-CXCL10 treatment resulted in increased {beta} cell mass in a spontaneous diabetes model in NOD mice as well. Furthermore, we found that the NOD mouse-derived {beta} cell lineage expressed CXCR3, and proliferation of CXCR3 gene-transduced {beta} cell line was inhibited by CXCL10 treatment in vitro (Y. Kanazawa, T. Shigihara, S. Narumi, and A. Shimada, unpublished observation). According to these findings, both in vivo- and in vitro-enhanced {beta} cell proliferation seemed to be due to the direct effect of CXCL10 neutralization, although more detailed studies are required to clarify this. Moreover, these results of the present study in which enhanced {beta} cell proliferation by CXCL10 neutralization maintained the {beta} cell mass, resulting in suppression of the onset of diabetes, are consistent with recent reports by others that replication of differentiated {beta} cells is essential to maintain the {beta} cell mass (43, 44). Not only in type 1 diabetes, but also in type 2 diabetes, it has been suggested that a reduction in {beta} cell mass is one of the most important factors in disease progression; therefore, CXCL10 neutralization may be useful for maintaining the {beta} cell mass in both types of diabetes.

In conclusion, the present study demonstrated that CXCL10 neutralization by pCAGGS-CXCL10 plasmid DNA treatment prevented spontaneous diabetes through enhancement of {beta} cell proliferation in NOD mice. Cytokine/chemokine plasmid DNA "vaccination" is an easy, less invasive, and useful way to neutralize an encoded cytokine/chemokine for a long period, instead of frequent Ab injections. At the present time, some trials in human type 1 diabetes such as the Diabetes Prevention Trial-Type 1 diabetes (45) and the European Nicotinamide Diabetes Intervention Trial (46) have been unsuccessful, and anti-CD3 mAb therapy could not induce remission in newly onset type 1 diabetic patients (47). In this situation, we propose combination therapy of CXCL10 neutralization and an immunomodulator such as insulin and/or anti-CD3 Ab because there is a possibility to prevent or cure human type 1 diabetes by promotion of "replication" of {beta} cells and immunomodulation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was partly supported by a Grant-in-Aid for Young Scientists (B) (to T. Shigihara), the Keio Gijuku Fukuzawa Memorial Fund for the Advancement of Education and Research, and the Nateglinide Memorial Toyoshima Research and Education Fund. Back

2 Address correspondence and reprint request to Dr. Akira Shimada at the current address: Department of Internal Medicine, Division of Endocrinology and Metabolism, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail address: asmd{at}sc.itc.keio.ac.jp Back

3 Abbreviations used in this paper: CY, cyclophosphamide; pLN, pancreatic lymph node; GAD, glutamic acid decarboxylase; PDX-1, pancreatic duodenal homeobox-1. Back

Received for publication May 3, 2005. Accepted for publication October 11, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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