Female Non-obese Diabetic Mice Research Articles

The specific p38 mitogen-activated protein kinase pathway inhibitor FR167653 keeps insulitis benign in nonobese diabetic mice

The p38 mitogen-activated protein kinase (MAPK) pathway is important in Th1 immunity, macrophage activation, and apoptosis. Since they may be associated with β-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 β-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-γ 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.

Autoantibodies and CD4 T cells target a beta cell retroviral envelope protein in non-obese diabetic mice.

We determined that, over a biologic time interval, from 4 to 8 weeks of age, female non-obese diabetic (NOD) mice develop antibodies against pancreatic beta-cell-surface antigens depending upon the presence of both the MHC class II susceptibility allele, I-A(g7), and other NOD background genes. We generated a mAb from a pre-diabetic NOD mouse that binds to the surface of insulinoma cells and isolated mouse beta cells, and identified the target as a retroviral envelope glycoprotein expressed on pancreatic beta cells. The cloned and expressed sequence for this protein was recognized by the mAb. The antibody as well as sera from pre-diabetic NOD mice recognized the recombinant protein. Spontaneous T cell reactivity against a peptide from the cloned protein was found in NOD mice. In conclusion, a beta cell retroviral envelope protein is a target antigen that is selected by the NOD mouse immune system early in the pathogenesis of autoimmune diabetes.

Local adeno-associated virus-mediated interleukin 10 gene transfer has disease-modifying effects in a murine model of Sjögren's syndrome.

Female nonobese diabetic (NOD) mice develop spontaneous autoimmune sialadenitis and loss of salivary flow, and are a widely used model of Sjögren's syndrome. We examined the feasibility of local salivary gland immunomodulatory gene delivery to alter these sequelae in NOD mice. We constructed recombinant adeno-associated virus (rAAV) vectors encoding either human interleukin 10 (rAAVhIL-10) or beta-galactosidase (rAAVLacZ, control vector). Mice received rAAVhIL-10 or rAAVLacZ by retrograde submandibular ductal instillation either at age 8 weeks (early, before onset of sialadenitis), or at 16 weeks (late, after onset of sialadenitis). As a systemic treatment control, separate mice received intramuscular delivery of rAAVhIL-10 at each time point. Both submandibular and intramuscular delivery of vector led to low circulating levels of hIL-10. After submandibular administration of rAAVhIL-10, salivary flow rates at 20 weeks for both the early and late treatment groups were significantly higher than for both rAAVLacZ-administered and untreated mice. Systemic delivery of rAAVhIL-10 led to improved salivary flow in the late treatment group. Inflammatory infiltrates in submandibular glands, however, were significantly reduced only in mice receiving rAAVhIL-10 locally in the salivary gland compared with mice receiving this vector intramuscularly, or rAAVLacZ or no treatment. In addition, after submandibular rAAVhIL-10 delivery, NOD mice exhibited significantly lower blood glucose, and higher serum insulin, levels than all other groups, indicating some systemic benefit of this treatment. These studies show that expression of hIL-10 by rAAV vectors can have disease-modifying effects in the salivary glands of NOD mice, and suggest that local immunomodulatory gene transfer may be useful for managing the salivary gland pathology in Sjögren's syndrome.

Immunization of non-obese diabetic (NOD) mice with glutamic acid decarboxylase-derived peptide 524-543 reduces cyclophosphamide-accelerated diabetes.

NOD mice constitute a model for studying the prevention of human autoimmune type 1 diabetes. Glutamic acid decarboxylase (GAD) could be a key antigen involved in this disease, and GAD65 peptide 524-543 has been implicated in early T cell response in young NOD mice. We performed two i.p. injections of GAD peptide 524-543 (100 micrograms at each injection), together with Freund's incomplete adjuvant (FIA), into female NOD mice at 30 and 45 days old. Diabetes was accelerated 2 weeks later by a single injection of cyclophosphamide (CY), which acts against suppressive mechanisms. Treatment with GAD 524-543 peptide delayed the onset of diabetes and reduced its incidence (28% versus 60%; P < 0.001) compared with control mice injected with FIA alone, or GAD peptide 534-553, or an irrelevant peptide. In the same group, the severity of lymphocytic inflammation of pancreatic islets was reduced (P < 0.03). Up to 3 months after peptide injections, a strong splenocytic proliferative response occurred in immunized NOD mice against the immunizing peptide alone (but not against a panel of seven other GAD65-derived peptides). After peptide challenge of splenocytes in vitro, protection against CY-accelerated diabetes was associated with higher peptide-specific production of T helper type 2 (Th2)-associated interleukins 4 and 10, whereas Th1-associated interferon-gamma and IL-2 were proportionally less represented. During contransfer, T splenocytes from GAD 524-543-immunized mice were able to reduce the capacity of T cells from diabetic donors to transfer the disease adoptively (P < 0.01), demonstrating the generation of cellular mechanisms that actively suppress the disease. It is concluded that immunization of NOD mice with GAD65 peptide 524-543 can counteract CY-accelerated diabetes, possibly through active cellular suppression linked to a shift of Th1/Th2 balance toward the production of Th2 cytokines such as IL-4 and IL-10. This study provides additional support for the notion that GAD, and more precisely its epitope 524-543, could be one of the key targets for the pathogenesis of type 1 diabetes in NOD mice, as well as for the efficacy of disease-specific peptide therapy in type 1 diabetes.

IL-1 receptor antagonist inhibits recurrence of disease after syngeneic pancreatic islet transplantation to spontaneously diabetic non-obese diabetic (NOD) mice.

The effect of an IL-1 receptor antagonist on recurrence of hyperglycaemia after syngeneic pancreatic islet transplantation to spontaneously diabetic female NOD mice was investigated. The transplanted animals were treated with either the receptor antagonist (8.0 mg/kg body weight per day for 12-14 days) or PBS, delivered by subcutaneously implanted osmotic pumps. In the control animals, a transient normoglycaemia was achieved, but hyperglycaemia was generally observed 6 days after islet transplantation. Administration of IL-1 receptor antagonist had a clear protective effect against recurrence of hyperglycaemia until day 14, but after cessation of drug delivery hyperglycaemia re-appeared. The results indicate that continuous administration of the IL-1 receptor antagonist can prevent recurrence of the diabetogenic process in NOD mice. IL-1 receptor antagonist may therefore become a useful adjuvant immunomodulating therapy after human islet transplantation in insulin-dependent diabetes mellitus.

Effects of two pediatric vaccines on autoimmune diabetes in NOD female mice

The induction or exacerbation of autoimmune diseases is a potential adverse effect of immunostimulating drugs. Vaccines have been suspected of such actions. Epidemiological studies, however, have so far failed to demonstrate any causal relationship between vaccination and autoimmune diseases, including insulin-dependent diabetes mellitus (IDDM). In this study, autoimmune diabetes-prone non-obese diabetic (NOD) mice were treated with two multivalent diphtheria, tetanus, pertussis, poliomyelitis and haemophilus vaccines (diphtheria, tetanus, acellular pertussis, inactivated polio (DTaP-IVP) or DTaP-IVP/Haemophilus influenza type b (Hib)) intraperitoneally at each of 10, 12 and 14 weeks of age. Although non-statistically significant, the incidence of autoimmune diabetes was slightly reduced by the DTaP-IVP vaccine. Blood glucose levels were actually significantly reduced in the mice treated with the DTaP-IVP vaccine relative to the untreated control mice. A slight decrease in blood glucose levels amongst the mice given the DTaP-IVP/Hib vaccine was also noted. Therefore this study does not support previous claims that children’s vaccination might be associated with acceleration or exacerbation of IDDM.

Systemic overexpression of IL-10 induces CD4+CD25+ cell populations in vivo and ameliorates type 1 diabetes in nonobese diabetic mice in a dose-dependent fashion.

Early systemic treatment of nonobese diabetic mice with high doses of recombinant adeno-associated virus (rAAV) vector expressing murine IL-10 prevents type 1 diabetes. To determine the therapeutic parameters and immunological mechanisms underlying this observation, female nonobese diabetic mice at 4, 8, and 12 wk of age were given a single i.m. injection of rAAV-murine IL-10 (10(4), 10(6), 10(8), and 10(9) infectious units (IU)), rAAV-vector expressing truncated murine IL-10 fragment (10(9) IU), or saline. Transduction with rAAV-IL-10 at 10(9) IU completely prevented diabetes in all animals injected at all time points, including, surprisingly, 12-wk-old animals. Treatment with 10(8) IU provided no protection in the 12-wk-old injected mice, partial prevention in 8-wk-old mice, and full protection in all animals injected at 4 wk of age. All other treatment groups developed diabetes at a similar rate. The rAAV-IL-10 therapy attenuated pancreatic insulitis, decreased MHC II expression on CD11b+ cells, increased the population of CD11b+ cells, and modulated insulin autoantibody production. Interestingly, rAAV-IL-10 therapy dramatically increased the percentage of CD4+CD25+ regulatory T cells. Adoptive transfer studies suggest that rAAV-IL-10 treatment alters the capacity of splenocytes to impart type 1 diabetes in recipient animals. This study indicates the potential for immunomodulatory gene therapy to prevent autoimmune diseases, including type 1 diabetes, and implicates IL-10 as a molecule capable of increasing the percentages of regulatory cells in vivo.

Suppressive effects of a selective inducible nitric oxide synthase (iNOS) inhibitor on pancreatic beta-cell dysfunction.

Type 1 diabetes mellitus is an autoimmune disease characterized by dysfunction and destruction of the pancreatic beta cells. Interleukin-1beta (IL-1beta) has been reported to cause suppression of insulin secretion from pancreatic islets via induction of inducible nitric oxide synthase (iNOS) followed by nitric oxide (NO) production. In this study, we investigated the effects of inhibition of iNOS on pancreatic beta-cell dysfunction in non-obese diabetic (NOD) mice and IL-1beta-treated isolated rat pancreatic islets using a novel specific inhibitor, ONO-1714. Female NOD mice which received subcutaneous infusion of ONO-1714 (4 microg/kg/day or 40 microg/kg/day) from 10 to 14 weeks after birth were compared with untreated NOD mice. In addition, pancreatic islets were isolated from Sprague-Dawley rats and cultured for 24 h with IL-1beta (100 U/mL) with or without ONO-1714 or the non-selective NOS inhibitor NG-monomethyl-L-arginine (L-NMMA). We measured insulin secretion and insulin content of the islets by ELISA, iNOS mRNA expression by reverse transcription-polymerase chain reaction, and NO generation by Griess Reagent System. Hyperglycaemia was observed in NOD mice. ONO-1714 treatment blunted this increase and tended to preserve insulin secretion, although body weight increase did not differ between the groups. Insulitis was also attenuated in the ONO-1714-administered group compared to the control group. Furthermore, in isolated rat pancreatic islets ONO-1714 prevented IL-1beta-induced inhibition of insulin secretion, this protection being evident in much lower concentrations than with L-NMMA. While ONO-1714 completely inhibited IL-1beta-induced NO production, it did not reduce expression of islet iNOS mRNA. These findings indicate that ONO-1714 is promising as a therapeutic agent for autoimmune diabetes.

The programmed death-1 (PD-1) pathway regulates autoimmune diabetes in nonobese diabetic (NOD) mice.

Programmed death-1 (PD-1) receptor, an inhibitory costimulatory molecule found on activated T cells, has been demonstrated to play a role in the regulation of immune responses and peripheral tolerance. We investigated the role of this pathway in the development of autoimmune diabetes. PD-1 or PD-L1 but not PD-L2 blockade rapidly precipitated diabetes in prediabetic female nonobese diabetic (NOD) mice regardless of age (from 1 to 10-wk-old), although it was most pronounced in the older mice. By contrast, cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) blockade induced disease only in neonates. Male NOD mice also developed diabetes after PD-1–PD-L1 pathway blockade, but NOR mice, congenic to NOD but resistant to the development of diabetes, did not. Insulitis scores were significantly higher and frequency of interferon γ–producing GAD-reactive splenocytes was increased after PD-1–PD-L1 pathway blockade compared with controls. Interestingly, PD-L1 but not PD-L2 was found to be expressed on inflamed islets of NOD mice. These data demonstrate a central role for PD-1–PD-L1 interaction in the regulation of induction and progression of autoimmune diabetes in the NOD mouse and provide the rationale to develop new therapies to target this costimulatory pathway in this disease.

Immunohistochemical Demonstration of Leptin in Pancreatic Islets of Non-Obese Diabetic and CD-1 mice: Co-localization in Glucagon Cells and its Attenuation at the Onset of Diabetes

Leptin is a 16 kD polypeptide hormone produced predominantly by white adipose tissue and exerts profound effects on food intake and energy balance. More recent studies have shown extra sites of leptin production in human and rodent tissues and have ascribed additional roles for the hormone, e.g., in immune and reproductive functions. A role for the hormone has also been implicated in insulin-dependent diabetes mellitus in the non-obese diabetic (NOD) mouse. However, whether leptin originates from islet cells of the mouse is not known. Here dual-label immunohistochemistry was employed to examine leptin expression in islet cells, and its distribution and cellular sources in pancreatic sections of female NOD/Ak and CD-1 mice of various ages. For comparison, leptin immunolabelling was examined in adult pancreatic sections from male NOD/Ak CD-1, Balb/c and FVB/N mice and female severe combined immunodeficient CB. 17 mice. Pancreatic tissues from adult female guinea pig, sheep and cattle and neonatal pigs were also studied. Our results show that in the day 1 NOD and CD-1 mice, leptin immunolabelling was observed in selective glucagon cells within the developing islets while at days 15 and 22, it became more intense and co-incident. This pattern of staining was maintained at days 40, 90, 150 and 250. In the female NOD mouse, leptin was absent in intra-islet immune cells. Its expression was variable in islets from male NOD and CD-1 mice. In spontaneously diabetic female NOD mice and following acceleration of diabetes with cyclophosphamide, despite the persistence of strong immunolabelling for glucagon in the re-distributed alpha cells, leptin expression was either absent, diminished or present in only a proportion of alpha cells. The reduction in leptin labelling was often associated with diabetic islets which had insulitis in association with only a small number of residual beta cells. Leptin expression was absent in guinea pig, ovine, bovine and neonatal porcine islet cells, despite the expression of intensely labelled glucagon cells. The present results demonstrate leptin co-localization in glucagon cells of the mouse islet. Its expression diminishes in the presence of inadequate insulin. Leptin produced within the mouse islet may have bi-directional influences on leptin and insulin regulation and may play local functions in islet development and metabolism.

High-level expression of interleukin-4 following electroporation-mediated gene transfer accelerates Type 1 diabetes in NOD mice

Nonobese diabetic (NOD) mice develop T cell-dependent autoimmune disease. Administration of interleukin-4 (IL-4), one of the T helper 2 (Th2) cytokines, is reported to prevent either insulitis or diabetes or both in NOD mice. We examined the effect of transferring an IL-4-expressing plasmid vector into muscle by in vivo electroporation on the progression of diabetes in NOD mice. Plasmid DNA expressing murine IL-4 (pCAGGS-IL-4) was introduced into the muscles of 4- and 6-week-old female NOD mice using an in vivo electroporation technique we developed previously. The serum IL-4 levels reached 2000–8000 pg/ml 3 days after the delivery of pCAGGS-IL-4 and remained detectable (>5 pg/ml) for over 4 weeks. In contrast to the previous reports, 88% of the mice treated with pCAGGS-IL-4 developed overt diabetes by 30 weeks of age, while only 25% of nontreated mice and 19% of the mice treated with control pCAGGS developed overt diabetes by then ( p<0.01). Therefore, highly expressed IL-4 introduced by in vivo electroporation may have caused a Th1 shift, resulting in the promotion of diabetes in NOD mice. The high serum concentration of cytokines attained by our method is likely to unveil previously unknown cytokine functions.

Characterization of antibody responses to endogenous and exogenous antigen in the nonobese diabetic mouse

It is suggested that a T-helper cell 2 (Th2) shift and Th2 spreading of autoimmunity following immunization with beta-cell antigen causes diabetes protection. To address this, antibody titer and subclass to insulin, glutamic acid decarboxylase (GAD)65, IA-2, and IA-2β proteins were measured by radiobinding assays in untreated or immunized female nonobese diabetic mice. Untreated nonobese diabetic mice developed autoantibodies to insulin (IAA), but not GAD or IA-2/IA-2β, and IAA-positive mice had increased diabetes risk ( P < 0.001). IAA were IgG1 and IgG2b. In immunized mice, IgG1 and lesser IgG2b insulin antibodies were promoted by subcutaneous injection of insulin plus incomplete Freund’s adjuvant, insulin plus Montanide ISA 720, and glucagon plus incomplete Freund’s adjuvant, but not by incomplete Freund’s adjuvant plus GAD65, IA-2β, or phenylethanolamine N-methyltransferase, or adjuvant alone. Diabetes incidence was significantly reduced in immunized groups with elevated insulin antibody (IA) responses. Spreading of antibody responses to GAD or IA-2/IA-2β following immunization was rare, and antibody epitope spreading was only detected in IA-2β immunized mice. Humoral autoimmunity in nonobese diabetic mice is, therefore, limited to IAA with Th2 subclass phenotype and is associated with increased diabetes risk. This contrasts the diabetes protection provided by immunization protocols that promote this response and suggests that Th2 immunity may not be the principal regulator of beta-cell destruction in autoimmune diabetes.

Assessment of β Cell Mass and Oxidative Peritoneal Exudate Cells in Murine Type 1 Diabetes using Adoptive Transfer System

Because it is controversial how the g cell mass is reduced during the disease process in type 1 diabetes, we transferred splenocytes from Non-obese diabetic (NOD) to NOD-scid mice and evaluated the relation between the status of the pancreas in donors and the time taken to transfer diabetes to the recipients. We also evaluated the usefulness of assessment of the proportion of oxidative peritoneal exudate cells (PEC) as a novel marker of disease activity in this system. We examined the proportion of oxidative PEC, pancreatic insulin content and pancreatic histology in 16-18-week-old female NOD mice (donors), and transferred their splenocytes into 5-week-old female NOD-scid mice (recipients). After the onset of diabetes in NOD-scid recipients, we assessed the relation between insulin content (or severity of insulitis) of NOD donors and the time taken to transfer diabetes to NOD-scid recipients. The insulin content of "diabetes-prone" donors whose disease status was considered to be just before the onset of diabetes ("malignant" donors) was the same as that of diabetic mice, whereas the insulin content of "diabetes-prone" donors excluding "malignant" donors ("benign" donors) was the same as that of "non-diabetes-prone" donors. Because its proportion of oxidative PEC was inversely correlated with the severity of insulitis, we then evaluated the relation between the proportion of oxidative PEC and the time taken to transfer diabetes. "Malignant" donors had less proportion of oxidative PEC (<10%), as compared to "benign" and "non-diabetes-prone" donors. These results suggest that a marked reduction of g cell mass occurs at the very late prediabetic stage, and assessment of the proportion of oxidative PEC is useful to evaluate disease activity in type 1 diabetes.

Kinetics of TNF-alpha and IFN-gamma mRNA expression in islets and spleen of NOD mice.

Insulin-dependent diabetes mellitus is caused by autoimmune destruction of pancreatic beta cells. Non-obese diabetic (NOD) mice spontaneously develop diabetes similar to the human disease. Cytokines produced by islet-infiltrating mononuclear cells may be directly cytotoxic and can be involved in islet destruction coordinated by CD4+ and CD8+ cells. We utilized a semiquantitative RT-PCR assay to analyze in vitro the mRNA expression of TNF-alpha and IFN-gamma cytokine genes in isolated islets (N = 100) and spleen cells (5 x 10(5) cells) from female NOD mice during the development of diabetes and from female CBA-j mice as a related control strain that does not develop diabetes. Cytokine mRNAs were measured at 2, 4, 8, 14 and 28 weeks of age from the onset of insulitis to the development of overt diabetes. An increase in IFN-gamma expression in islets was observed for females aged 28 weeks (149 +/- 29 arbitrary units (AU), P<0.05, Student t-test) with advanced destructive insulitis when compared with CBA-j mice, while TNF-alpha was expressed in both NOD and CBA-j female islets at the same level at all ages studied. In contrast, TNF-alpha in spleen was expressed at higher levels in NOD females at 14 weeks (99 +/- 8 AU, P<0.05) and 28 weeks (144 +/- 17 AU, P<0.05) of age when compared to CBA-j mice. The data suggest that IFN-gamma and TNF-alpha expression in pancreatic islets of female NOD mice is associated with beta cell destruction and overt diabetes.

Increased astrocyte reactivity in the hippocampus of murine models of type 1 diabetes: the nonobese diabetic (NOD) and streptozotocin-treated mice

Diabetes can be associated with cerebral dysfunction in humans and animal models of the disease. Moreover, brain anomalies and alterations of the neuroendocrine system are present in type 1 diabetes (T1D) animals, such as the spontaneous nonobese diabetic (NOD) mouse model and/or the pharmacological streptozotocin (STZ)-induced model. Because of the prevalent role of astrocytes in cerebral glucose metabolism and their intimate connection with neurones, we investigated hippocampal astrocyte alterations in prediabetic and diabetic NOD mice and STZ-treated diabetic mice. The number and cell area related to the glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes were quantified in the stratum radiatum region of the hippocampus by computerized image analysis in prediabetic (2, 4 and 8 weeks of age) and diabetic (16-week-old) NOD female mice, age and sex-matched lymphocyte-deficient NOD scid and C57BL/6 control mice and, finally, STZ-induced diabetic and vehicle-treated nondiabetic 16-week-old C57BL/6 female mice. Astrocyte number was higher early in life in prediabetic NOD and NOD scid mice than in controls, when transient hyperinsulinemia and low glycemia were found in these strains. The number and cell area of GFAP + cells further increased after the onset of diabetes in NOD mice. Similarly, in STZ-treated diabetic mice, the number of GFAP + cells and cell area were higher than in vehicle-treated mice. In conclusion, astrocyte changes present in genetic and pharmacological models of T1D appear to reflect an adaptive process to alterations of glucose homeostasis.

Role of stromal-cell derived factor-1 in the development of autoimmune diseases in non-obese diabetic mice.

The chemokine stromal-cell derived factor-1 (SDF-1) controls maturation, trafficking, and homing of certain subsets, lymphoid cells including immunogenic B and T cells, as a ligand of the CXCR4 chemokine receptor. Insulin-dependent diabetes mellitus (IDDM) and Sjögren's syndrome (SS), both highly regulated autoimmune diseases, develop spontaneously in non-obese diabetic (NOD) mice. To investigate the role of SDF-1 in the development of autoimmune diseases, we injected groups of NOD female mice with antibodies to SDF-1 (anti-SDF-1), which resulted in a 30% reduction of diabetes up to 30 weeks of age, delayed average diabetes onset by 10 weeks, and suppressed insulitis. Autoimmune sialoadenitis was evident in anti-SDF-1-injected mice (SDF-1-Ig group) at the same level as in all groups of mice, whether injected with non-specific antibodies or not. In addition, in the SDF-1-Ig group, a greater number of immunoglobulin M (IgM)- IgD- B220(low) CD38+ CD43+ CD23- progenitor B cells and IgM+ IgD+ B220(high) CD43- CD38+ CD24+ CD23+ mature B cells remained in the bone marrow, whereas infiltration of mature IgM+ B cells was less extensive in peripheral tissues. Our results suggested that anti-SDF-1 antibodies injection was effective in inhibiting diabetes and insulitis without affecting autoimmune sialoadenitis or SS in NOD mice. SDF-1 may be an essential chemokine for trafficking and migration of autoreactive B cells in the development of diabetes.

A murine interleukin-4-Ig fusion protein regulates the expression of Th1- and Th2-specific cytokines in the pancreas of NOD mice.

Interleukin (IL)-4 is a key cytokine in T-helper type 2 (Th2) immune response. We have constructed a dimeric IL-4 molecule consisting of the murine IL-4 and the murine Fc part of IgG2a. We first tested the biological activity of the chimeric protein by in vitro studies using isolated murine spleen cells. IL-4-Ig was found to downregulate LPS-induced IFN-gamma and TNF-alpha production. The immunomodulatory potential of the fusion protein was also analyzed in non-obese diabetic (NOD) mice, an animal model for human type 1 diabetes. Female NOD mice aged 10 weeks were treated once with cyclophosphamide to accelerate and synchronize the progression of insulitis. Treatment with IL-4-Ig induced strong modulation of the pancreatic cytokine balance. Expression was downregulated for both Th1-specific cytokine IFN-gamma and the Th2-specific cytokine IL-10. IL-12p40 expression was only slightly affected. Interestingly, infiltration increased in the islets of IL-4-Ig-treated animals, and therefore did not correlate with the decreased IFN-gamma expression. Hence, IL-4-Ig did not prevent the progression from peri- to intra-insulitis, but suppressed inflammatory cytokine production. In most experiments, the biological activity of IL-4-Ig and IL-4 was comparable. We conclude that treatment with the chimeric protein IL-4-Ig effectively downregulates Th1 responses but simultaneously augments intra-islet infiltration.

Polyethylene glycolated recombinant TNF receptor I improves insulitis and reduces incidence of spontaneous and cyclophosphamide-accelerated diabetes in nonobese diabetic mice.

We have conducted three studies to examine the role of TNFalpha in islet destruction in female nonobese diabetic mouse (NOD) mice, a model of human autoimmune diabetes, using polyethylene glycolated (PEGylated) soluble TNF receptor type I (PEG sTNF-RI) as TNFalpha antagonist. PEG sTNF-RI (3 mg/kg, sc) was given every other day to NOD mice from age wk 8 for 12 wk (study 1), from age wk 12 for 8 wk (study 2), or from age wk 8 for 3 wk, with cyclophosphamide (6 mg/mouse) injected at wk 9 to accelerate the onset of diabetes (study 3). Diabetic incidence was reduced (control vs. PEG sTNF-RI) from 68.7% (11 of 16) to 18.3% (3 of 16) in study 1, from 84.6% (11 of 13) to 28.5% (4 of 14) in study 2, and from 66.6% (8 of 12) to 23.1% (3 of 13) in study 3, respectively. The incidence of insulitis was also reduced from 91.6% (11 of 12) to 12.5% (2 of 16) in study 1 and from 100% (7 of 7) to 16.6% (2 of 12) in study 2 by PEG sTNF-RI. PEG sTNF-RI also largely preserved islet insulin content, reduced mRNA of inducible nitric oxide synthase and IL-6 in pancreases, and lowered plasma corticosterone, glycerol, and free fatty acid levels. These results confirm a pathogenic role of TNFalpha in mediating insulitis in NOD mice and suggest the prophylactic and therapeutic potential of PEG sTNF-RI for human autoimmune diabetes.

Nondepleting anti-CD4 and soluble interleukin-1 receptor prevent autoimmune destruction of syngeneic islet grafts in diabetic NOD mice.

Successful islet transplantation in type 1 diabetes requires tolerance induction of both allo- and autoreactive T-cell responses. Monoclonal antibodies targeting the CD4 coreceptor on T-helper cells have been shown to be effective in this regard. In type 1 diabetes, there is some evidence to suggest that cytokines such as interleukin (IL)-1 may be involved in beta-cell destruction. The high glucose levels associated with type 1 diabetes are also known to be toxic to beta cells. The tempo of T-cell and macrophage infiltration into syngeneic islets transplanted into diabetic nonobese diabetic (NOD) mice was examined by immunohistochemistry. We investigated the ability of a nondepleting anti-CD4 monoclonal antibody (YTS177) to induce tolerance to syngeneic islet grafts in female spontaneous diabetic NOD mice and in an adoptive transfer model of diabetes in NOD mice. The spontaneous model was used to test the effect on graft function of perioperative insulin therapy in mice treated with YTS177. The ability of soluble interleukin (sIL)-1 receptor (R) type II (sIL-1RII) to inhibit IL-1 effects in syngeneic islet transplants was also assessed. Cellular infiltration of CD3 cells and macrophages into the islet graft coincided with loss of graft function in untreated mice. Self-tolerance to beta cells was restored with YTS177, allowing long-term graft survival in a proportion of animals. The use of perioperative insulin therapy increased the number of successful grafts in spontaneously diabetic NOD mice treated with YTS177. The combination of YTS177 with sIL-1RII significantly improved the rates of graft survival compared with graft survival in YTS177-treated spontaneously diabetic NOD mice. Nondepleting anti-CD4 antibodies restore self tolerance to syngeneic islet transplants in diabetic NOD mice. Insulin therapy improves graft survival in mice treated with YTS177. Preventing the action of IL-1 greatly improves graft survival induced with YTS177.

T cell response to preproinsulin I and II in the nonobese diabetic mouse.

Immunization against insulin, insulin B chain, or B chain peptide B(9-23) (preproinsulin peptide II(33-47)) prevents diabetes in the nonobese diabetic (NOD) mouse. Whether or not peptide II(33-47) is the only proinsulin determinant recognized by CD4 T cells remains unclear. Using two peptide libraries spanning the entire sequence of preproinsulin I and preproinsulin II, respectively, we identified T cells specific for four proinsulin epitopes within the islet cell infiltrate of prediabetic female NOD mice. These epitopes were among immunogenic epitopes to which a T cell response was detected after immunization of NOD mice with individual peptides in CFA. Immunogenic epitopes were found on both isoforms of insulin, especially proinsulin II, which is the isoform expressed in the thymus. The autoimmune response to proinsulin represented only part of the immune response to islet cells within the islet cell infiltrate in 15-wk-old NOD mice. This is the first systematic study of preproinsulin T cell epitopes in the NOD mouse model.