Intrathymic islet cell transplantation reduces beta-cell autoimmunity and prevents diabetes in NOD/Lt mice.

Intrathymic transplantation of syngeneic islets into adolescent NOD/Lt mice was performed to establish whether the thymus would serve as an immunoprivileged site for beta-cell engraftment, and whether this treatment would prevent the development of diabetes by eliciting tolerance to islet antigens. Intrathymic injection of cells from 200 NOD islets into 4-wk-old female NOD/Lt mice produced a significant reduction in the severity of insulitis at 24 wk of age. Furthermore, diabetes development was strongly suppressed (11% incidence) compared with controls (100% incidence). Both thymus histology and thymic insulin content revealed a rapid loss of the implanted beta-cells with < 1% remaining 1 wk posttransplantation. Despite the rapid loss of thymus-implanted islet cells, evidence for tolerance induction to islet cell antigens was obtained by adoptive transfer of splenic leukocytes from these mice into NOD-scid/scid recipients. After adoptive transfer of splenic leukocytes from 24-wk-old untreated prediabetic donors, 4 of 5 NOD-scid/scid recipients developed diabetes within 4 wk, and none of the recipients became diabetic after transfer of splenocytes from intrathymic islet-implanted donors. Intrathymic islet transplantation did not lead to reduction of sialitis in females with reduced severity of insulitis, indicating that the protective effect was tissue specific. This also was reflected in adoptive transfer experiments, because equal severity of sialitis was observed in NOD-scid/scid recipients of spleen cells from either islet transplanted or control NOD/Lt mice. In conclusion, the data suggest that intrathymic injection of islet cells prevents diabetes by stimulating immunological tolerance to beta-cells.     

Autoimmune diabetes and resistance to xenograft transplantation tolerance in NOD mice

Costimulation blockade induces prolonged rat islet and skin xenograft survival in C57BL/6 mice. Nonobese diabetic (NOD) mice, which are used to model human autoimmune diabetes, are resistant to costimulation blockade-induced allograft tolerance. We tested the hypothesis that NOD mice would also be resistant to costimulation blockade-induced rat xenograft tolerance. We report that rat islet xenograft survival is short in spontaneously diabetic NOD mice treated with a tolerizing regimen of donor-specific transfusion and anti-CD154 antibody. Rat islet xenograft survival is only marginally longer in chemically diabetic NOD mice treated with costimulation blockade but is prolonged further in NOD Idd congenic mice bearing C57-derived chromosome 3 loci. Reciprocally, the presence of NOD-derived chromosome 3 loci shortens islet xenograft survival in tolerized C57BL/6 mice. Islet xenograft survival is longer in tolerized NOD.CD4a(-/-) and (NOD x C57BL/6)F1 mice than in NOD mice but still much shorter than in C57BL/6 mice. Skin xenograft survival in (NOD x C57BL/6)F1 mice treated with costimulation blockade is short, suggesting a strong genetic resistance to skin xenograft tolerance induction. We conclude that the resistance of NOD mice to xenograft tolerance induction involves some mechanisms that also participate in the expression of autoimmunity and other mechanisms that are distinct.     

Flt3-ligand treatment prevents diabetes in NOD mice

The mechanism by which mixed chimerism reverses autoimmunity in type 1 diabetes has not been defined. NOD mice have a well-characterized defect in the production of myeloid progenitors that is believed to contribute significantly to the autoimmune process. We therefore investigated whether chimerism induces a correction of this defect. Mixed chimerism restored production of myeloid progenitors in NOD mice to normal levels. Notably, NOD bone marrow cells as well as donor bone marrow cells produced the mature myeloid progeny, and the level of donor chimerism was not correlated with the degree of restoration of the defect. Moreover, NOD bone marrow cells cultured with Flt3-ligand developed a heat-stable antigen-positive/Ly6C+ population comprised primarily of mature myeloid dendritic cells, suggesting that the underlying abnormality is not cell intrinsic but rather due to a block in development of mature myeloid progeny, including myeloid dendritic cells. Strikingly, treatment of NOD mice with Flt3-ligand significantly decreased insulitis and progression to diabetes and was associated with a significant increase in myeloid dendritic cells and in vivo induction of CD4+/CD25+ cells in the pancreatic lymph node. Therefore, Flt3-ligand treatment and/or the establishment of mixed chimerism in prediabetic candidates may provide a benign and novel approach to treat diabetes.     

RegII is a beta-cell protein and autoantigen in diabetes of NOD mice

The Reg family of proteins has been studied in the context of growth and regeneration in several organs including pancreatic islets. We previously suggested that Reg proteins act as autoantigens in type 1 diabetes, based on evidence that a member of the Reg family (hepatocellular carcinoma intestine pancreas [HIP]/pancreatitis-associated protein [PAP]) was overexpressed in the islets of a patient who died after sudden onset of type 1 diabetes, and that, in NOD mice, Reg-specific T-cells adoptively transferred diabetes. In the current study, we developed antisera to detect individual Reg members in mouse islets and found that RegIIIalpha was present in the non-beta-cell portion of the islets, while RegII was predominantly expressed in beta-cells. Vaccination of NOD mice with the separately expressed N-terminal (NtfrII) or C-terminal (CtfrII) portion of RegII revealed a dichotomy: NtfrII vaccination accelerated and CtfrII vaccination delayed type 1 diabetes. Vaccination with CtfrII was more effective when given at later stages in the pathogenesis of type 1 diabetes, a time dependency different from that seen with other antigen-dependent vaccine strategies in NOD mice, which might have therapeutic implications. In conclusion, RegII is a novel beta-cell-derived autoantigen in NOD mice. The autoimmune response against this protein may convert a regenerative into an islet-destructive process accelerating development of type 1 diabetes.     

Interleukin-13 prevents autoimmune diabetes in NOD mice.

Interleukin (IL)-13 is a cytokine primarily produced by the T-helper (Th)-2 subset of lymphocytes that possesses powerful anti-inflammatory properties. Here, we have evaluated the impact of IL-13 treatment on development of type 1 diabetes in diabetes-prone nonobese diabetic (NOD) mice. Prolonged treatment with recombinant human IL-13 (hIL-13) markedly diminished the incidence of spontaneous type 1 diabetes in the mice. Female NOD mice treated from age 5-16 weeks with hIL-13 also showed significantly milder insulitis than control mice. The preventive action of hIL-13 was associated with a slight but significant change from a type 1 to a type 2 cytokine response. Accordingly, splenic lymphoid cells (SLC) from hIL-13-treated mice secreted less interferon (IFN)-gamma upon ex vivo stimulation with Concanavalin A than controls, and anti-CD3 monoclonal antibody-induced activation of T-cells in vivo resulted in lower blood levels of IFN-gamma and tumor necrosis factor-alpha and augmented blood levels of IL-4 in NOD mice pretreated with hIL-13. hIL-13 treatment also increased the blood levels of IgE and inhibited the transfer of type 1 diabetes by spleen cells from a diabetic donor to irradiated recipients. Taken together, these data add hIL-13 to the list of cytokines capable of downregulating immunoinflammatory diabetogenic pathways in NOD mice, and further support the concept that IL-4-related anti-inflammatory cytokines might have a role in the prevention of type 1 diabetes.     

Effects of autoimmunity and immune therapy on beta-cell turnover in type 1 diabetes

beta-Cell mass can expand in response to demand: during pregnancy, in the setting of insulin resistance, or after pancreatectomy. It is not known whether similar beta-cell hyperplasia occurs following immune therapy of autoimmune diabetes, but the clinical remission soon after diagnosis and the results of recent immune therapy studies suggest that beta-cell recovery is possible. We studied changes in beta-cell replication, mass, and apoptosis in NOD mice during progression to overt diabetes and following immune therapy with anti-CD3 monoclonal antibodies (mAbs) or immune regulatory T-cells (Tregs). beta-Cell replication increases in pre-diabetic mice, after adoptive transfer of diabetes with increasing islet inflammation but before an increase in blood glucose concentration or a significant decrease in beta-cell mass. The pathogenic cells are responsible for increasing beta-cell replication because replication was reduced during diabetes remission induced by anti-CD3 mAb or Tregs. beta-Cell replication stimulated by the initial inflammatory infiltrate results in increased production of new beta-cells after immune therapy and increased beta-cell area, but the majority of this increased beta-cell area represents regranulated beta-cells rather than newly produced cells. We conclude that beta-cell replication is closely linked to the islet inflammatory process. A significant proportion of degranulated beta-cells remain, at the time of diagnosis of diabetes, that can recover after metabolic correction of hyperglycemia. Correction of the beta-cell loss in type 1 diabetes will, therefore, require strategies that target both the immunologic and cellular mechanisms that destroy and maintain beta-cell mass.     

Systemic expression of heme oxygenase-1 ameliorates type 1 diabetes in NOD mice

Heme oxygenase-1 (HO-1) is an enzyme with potent immunoregulatory capacity. To evaluate the effect of HO-1 on autoimmune diabetes, female NOD mice at 9 weeks of age received a single intravenous injection of a recombinant adeno-associated virus bearing HO-1 gene (AAV-HO-1; 0.5 x 10(10)-2.5 x 10(10) viruses/mouse). In a dose-dependent manner, HO-1 transduction reduced destructive insulitis and the incidence of overt diabetes examined over a 15-week period. HO-1-mediated protection was associated with a lower type 1 T-helper cell (Th1)-mediated response. Adaptive transfer experiments in NOD.scid mice demonstrated that splenocytes isolated from AAV-HO-1-treated mice were less diabetogenic. Flow cytometry analysis revealed no significant difference in the percentages of CD4(+)CD25(+) regulatory T-cells between saline-treated and AAV-HO-1-treated groups. However, the CD11c(+) major histocompatibility complex II(+) dendritic cell population was much lower in the AAV-HO-1-treated group. A similar protective effect against diabetes was observed in NOD mice subjected to carbon monoxide (CO) gas (250 ppm CO for 2 h, twice per week). These data suggest that HO-1 slows the progression to overt diabetes in pre-diabetic NOD mice by downregulating the phenotypic maturity of dendritic cells and Th1 effector function. CO appears to mediate at least partly the beneficial effect of HO-1 in this disease setting.     

Glucagon receptor knockout prevents insulin-deficient type 1 diabetes in mice

OBJECTIVE

To determine the role of glucagon action in the metabolic phenotype of untreated insulin deficiency.

RESEARCH DESIGN AND METHODS

We compared pertinent clinical and metabolic parameters in glucagon receptor-null (Gcgr^−/−) mice and wild-type (Gcgr^+/+) controls after equivalent destruction of β-cells. We used a double dose of streptozotocin to maximize β-cell destruction.

RESULTS

Gcgr^+/+ mice became hyperglycemic (>500 mg/dL), hyperketonemic, polyuric, and cachectic and had to be killed after 6 weeks. Despite comparable β-cell destruction in Gcgr^−/− mice, none of the foregoing clinical or laboratory manifestations of diabetes appeared. There was marked α-cell hyperplasia and hyperglucagonemia (∼1,200 pg/mL), but hepatic phosphorylated cAMP response element binding protein and phosphoenolpyruvate carboxykinase mRNA were profoundly reduced compared with Gcgr^+/+ mice with diabetes—evidence that glucagon action had been effectively blocked. Fasting glucose levels and oral and intraperitoneal glucose tolerance tests were normal. Both fasting and nonfasting free fatty acid levels and nonfasting β-hydroxy butyrate levels were lower.

CONCLUSIONS

We conclude that blocking glucagon action prevents the deadly metabolic and clinical derangements of type 1 diabetic mice.The development of a radioimmunoassay for glucagon (1) established the hormonal status of this peptide (2), originally considered to be a contaminant of the insulin extraction process. Glucagon was immunocytochemically localized to pancreatic α-cells (3) and shown to be secreted in response to increased glucose need, as in starvation and exercise (4). By 1973, it was recognized that α-cell function was grossly abnormal in diabetes, particularly in type 1 diabetes (5). Here, β-cells are largely replaced by α-cells, and, without the inhibitory action of insulin, their secretion of glucagon is unrestrained, and glucagon action on the liver is unopposed. The result is a lethal hypercatabolic state. In 1973, the discovery of somatostatin (6), a potent inhibitor of glucagon secretion, made it possible to demonstrate the essential role of glucagon in the metabolic phenotype of type 1 diabetes (7–10).Those studies led to a search for a therapeutic suppressor of diabetic hyperglucagonemia or blocker of its action on the liver. In the 37 years since the discovery of somatostatin, only one other potent glucagon-suppressing substance, leptin, has been identified (11,12). By contrast, inactivators of glucagon have been less elusive. High affinity antiglucagon antibodies have benefited diabetic animals (13), as have a variety of molecules that block binding of glucagon to the glucagon receptor and/or block its signaling (14–18). Diabetic mice with glucagon receptor knockout (19) or mice treated with Gcgr antisense oligonucleotide similarly benefit from the elimination of glucagon action (20,21). Although all of the foregoing reports demonstrate that abrogation of glucagon action reduces hepatic overproduction of glucose, a potential therapeutic asset in diabetes management, none of the foregoing diabetic models studied thus far have been totally insulin-deficient, as in type 1 diabetes. In type 1 diabetes, islets are virtually devoid of β-cells, and are largely made up of hyperplastic α-cells. In contrast to normal α-cells, which are restrained by the high local concentrations of intraislet insulin (22), type 1 diabetic α-cells are unregulated, which results in inappropriate hyperglucagonemia (5). Moreover, with no insulin to oppose the hepatic actions of the hyperglucagonemia, unrestrained glycogenolysis, gluconeogenesis, ketogenesis, and hypercatabolism lead rapidly to ketoacidosis, cachexia, coma, and death.An essential role of hyperglucagonemia in the pathogenesis of this lethal syndrome has long been suspected but never fully proven. Recent studies of adenovirally induced hyperleptinemia in type 1 diabetic mice (12) indicate that glucagon suppression normalizes all metabolic parameters for more than a month despite a total absence of insulin. More recently, the same antidiabetic efficacy has been demonstrated with recombinant leptin (11,23). However, leptin-induced actions other than suppression of glucagon, such as increased insulin-like growth factor-1 (IGF-1) (12) and insulin-like growth factor-binding protein-2 (IGFBP-2) (23), may have contributed. To obtain unassailable proof that glucagon action by itself causes the lethal consequences of insulin deficiency, we induced insulin deficiency in glucagon receptor-null (Gcgr^−/−) mice. Gcgr^−/− mice were treated with streptozotocin (STZ), the most commonly used β-cytotoxins in rodents, in an effort to achieve complete insulin deficiency in the complete absence of glucagon activity. We compared the metabolic phenotype of complete β-cell destruction in mice in which glucagon action had been transgenically abrogated by knockout of the glucagon receptor (24). Because Gcgr^−/− mice are resistant to STZ-induced β-cell destruction (24), it was necessary to use a double dose of the β-cell poison STZ. Despite β-cell destruction equivalent to the wild-type (Gcgr^+/+) mice, Gcgr^−/− remained free of all manifestations of insulin deficiency.     

Fetal or neonatal low-glycotoxin environment prevents autoimmune diabetes in NOD mice

Advanced glycation end products (AGEs) are implicated in beta-cell oxidant stress. Diet-derived AGE (dAGE) are shown to contribute to end-organ toxicity attributed to diabetes. To assess the role of dAGE on type 1 diabetes, NOD mice were exposed to a high-AGE diet (H-AGE) and to a nutritionally similar diet with approximate fivefold-lower levels of N(epsilon)-carboxymethyllysine (CML) and methylglyoxal-derivatives (MG) (L-AGE). Suppression of serum CML and MG in L-AGE-fed mice was marked by suppression of diabetes (H-AGE mice >94% vs. L-AGE mice 33% in founder [F](0), 14% in F(1), and 13% in F(2) offspring, P < 0.006) and by a delay in disease onset (4-month lag). Survival for L-AGE mice was 76 vs. 0% after 44 weeks of H-AGE mice. Reduced insulitis in L-AGE versus H-AGE mice (P < 0.01) was marked by GAD- and insulin-unresponsive pancreatic interleukin (IL)-4-positive CD4+ cells compared with the GAD- and insulin-responsive interferon (IFN)-gamma-positive T-cells from H-AGE mice (P < 0.005). Splenocytes from L-AGE mice consisted of GAD- and insulin-responsive IL-10-positive CD4+ cells compared with the IFN-gamma-positive T-cells from H-AGE mice (P < 0.005). Therefore, high AGE intake may provide excess antigenic stimulus for T-cell-mediated diabetes or direct beta-cell injury in NOD mice; both processes are ameliorated by maternal or neonatal exposure to L-AGE nutrition.     

Role of beta-cells in type 1 diabetes pathogenesis

Whether autoimmunity results primarily from a defect of the immune system, target organ dysfunction, or both remains an open issue in most human autoimmune diseases. The highly multigenic background on which diabetes develops in the NOD mouse and in the human suggests that numerous gene variants associate in contributing to activation of autoimmunity to beta-cells. Both immune genes and islet-related genes are involved. The presence of beta-cells is required for initiation of diabetes autoimmunity to proceed. Available experiments in the NOD mouse and epidemiological evidence in the human point to proinsulin as a key autoantigen in diabetes. The functional importance of insulin, the high number of autoantigens characterized at different stages of diabetes, and their clustering within beta-cell subparticles point to the islet as a starting point in the initiation phase of the disease. Genes that direct the autoimmune reaction toward the beta-cell target, autoantigens that are recognized by autoreactive B- and T-cells along the autoimmune process, the importance of beta-cells in the activation of autoreactive lymphocytes, and the expression level of key beta-cell molecules along diabetes development are successively considered in this review.     

Synergistic reversal of type 1 diabetes in NOD mice with anti-CD3 and interleukin-1 blockade: evidence of improved immune regulation

Inflammatory cytokines are involved in autoimmune diabetes: among the most prominent is interleukin (IL)-1β. We postulated that blockade of IL-1β would modulate the effects of anti-CD3 monoclonal antibody (mAb) in treating diabetes in NOD mice. To test this, we treated hyperglycemic NOD mice with F(ab')(2) fragments of anti-CD3 mAb with or without IL-1 receptor antagonist (IL-1RA), or anti-IL-1β mAb. We studied the reversal of diabetes and effects of treatment on the immune system. Mice that received a combination of anti-CD3 mAb with IL-1RA showed a more rapid rate of remission of diabetes than mice treated with anti-CD3 mAb or IL-1RA alone. Combination-treated mice had increased IL-5, IL-4, and interferon (IFN)-γ levels in circulation. There were reduced pathogenic NOD-relevant V7 peptide-V7(+) T cells in the pancreatic lymph nodes. Their splenocytes secreted more IL-10, had increased arginase expression in macrophages and dendritic cells, and had delayed adoptive transfer of diabetes. After 1 month, there were increased concentrations of IgG1 isotype antibodies and reduced intrapancreatic expression of IFN-γ, IL-6, and IL-17 despite normal splenocyte cytokine secretion. These studies indicate that the combination of anti-CD3 mAb with IL-1RA is synergistic in reversal of diabetes through a combination of mechanisms. The combination causes persistent remission from islet inflammation.     

Administration of silica particles or anti-Lyt2 antibody prevents beta-cell destruction in NOD mice given cyclophosphamide

The cellular pathway of beta-cell destruction in type I (insulin-dependent) diabetes is still undefined. L3T4+ T-lymphocytes have a role in both the initiation of insulitis and in recurrent disease in transplanted allogeneic islets in nonobese diabetic (NOD) mice. The roles of macrophages and Lyt2+ T-lymphocytes in beta-cell destruction were studied in cyclophosphamide-induced diabetic NOD mice with silica particles and a rat anti-Lyt2 monoclonal antibody. After administration of cyclophosphamide, 10 of 26 untreated mice and 1 of 21 anti-Lyt2-treated mice became diabetic. Insulitis was significantly reduced in anti-Lyt2-treated mice, and immunocytochemical staining showed a lack of Lyt2+ cells. Only 1 of 19 silica-treated mice became diabetic, compared to 8 of 19 control mice. This study demonstrates that both Lyt2+ T-lymphocytes and macrophages are necessary, but not sufficient, for beta-cell destruction in NOD mice. Therefore, we propose that macrophages present beta-cell antigen to L3T4+ cells, which induce cytotoxic Lyt2+ cells to specifically destroy beta-cells.     

Autoantigen-specific B-cell depletion overcomes failed immune tolerance in type 1 diabetes

Eliminating autoantigen-specific B cells is an attractive alternative to global B-cell depletion for autoimmune disease treatment. To identify the potential for targeting a key autoimmune B-cell specificity in type 1 diabetes, insulin-binding B cells were tracked within a polyclonal repertoire using heavy chain B-cell receptor (BCR) transgenic (VH125Tg) mice. Insulin-specific B cells are rare in the periphery of nonautoimmune VH125Tg/C57BL/6 mice and WT/NOD autoimmune mice, whereas they clearly populate 1% of mature B-cell subsets in VH125Tg/NOD mice. Autoantigen upregulates CD86 in anti-insulin B cells, suggesting they are competent to interact with T cells. Endogenous insulin occupies anti-insulin BCR beginning with antigen commitment in bone marrow parenchyma, as identified by a second anti-insulin monoclonal antibody. Administration of this monoclonal antibody selectively eliminates insulin-reactive B cells in vivo and prevents disease in WT/NOD mice. Unexpectedly, developing B cells are less amenable to depletion, despite increased BCR sensitivity. These findings exemplify how a critical type 1 diabetes B-cell specificity escapes immune tolerance checkpoints. Disease liability is corrected by eliminating this B-cell specificity, providing proof of concept for a novel therapeutic approach for autoimmune disease.     

Costimulation blockade of both inducible costimulator and CD40 ligand induces dominant tolerance to islet allografts and prevents spontaneous autoimmune diabetes in the NOD mouse

Costimulation blockade is a promising strategy for preventing allograft rejection and inducing tolerance. Using a fully allogeneic mouse model, we tested the effectiveness of the combined blockade of the CD40 ligand and the inducible costimulator (ICOS) on islet allograft survival and in the prevention of autoimmune diabetes in the NOD mouse. Recipients treated with blocking monoclonal antibodies (mAbs) to ICOS and the CD40 ligand had significant prolongation of graft survival, with 26 of 28 functioning for >200 days. Long-term engrafted mice maintained antidonor proliferative and cytotoxic responses, but donor-specific immunization did not induce graft rejection, and challenge with second, same donor but not third-party grafts resulted in long-term acceptance. The immunohistology of tolerant grafts demonstrated the presence of CD4(+)CD25(+) T-cells expressing Foxp3, and islet/kidney composite grafts from tolerant mice, but not from mice lacking lymphocytes, were accepted indefinitely when transplanted into na?ve B6 mice, suggesting that recipient T-cells were necessary to generate dominant tolerance. Combined anti-ICOS and anti-CD40 ligand mAb therapy also prevented diabetes in NOD mice, with only 11% of treated recipients developing diabetes compared with 75% of controls. These data demonstrate that the blockade of CD40 ligand and ICOS signaling induces islet allograft tolerance involving a dominant mechanism associated with intragraft regulatory cells and prevents autoimmune diabetes in NOD mice.     

Blockade of tumor necrosis factor-related apoptosis-inducing ligand exacerbates type 1 diabetes in NOD mice

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is expressed in different tissues and cells, including pancreas and lymphocytes, and can induce apoptosis in various tumor cells but not in most normal cells. The specific roles of TRAIL in health and disease remain unclear. Here we show by cDNA array analyses that TRAIL gene expression is upregulated in pancreatic islets during the development of autoimmune type 1 diabetes in nonobese diabetic (NOD) mice and in Min6 islet beta-cells activated by TNF-alpha + interferon-gamma. However, stimulation of freshly isolated pancreatic islets or Min6 cells with TRAIL did not induce their apoptosis. TRAIL blockade exacerbates the onset of type 1 diabetes in NOD.Scid recipients of transferred diabetogenic T-cells and in cyclophosphamide-treated NOD mice. TRAIL inhibits the proliferation of NOD diabetogenic T-cells by suppressing interleukin (IL)-2 production and cell cycle progression, and this inhibition can be rescued in the presence of exogenous IL-2. cDNA array and Western blot analyses indicate that TRAIL upregulates the expression of the cdk inhibitor p27(kip1). Our data suggest that TRAIL is an important immune regulator of the development of type 1 diabetes.     

Minimal impact of a de novo-expressed beta-cell autoantigen on spontaneous diabetes development in NOD mice

During an autoimmune process, the autoaggressive response spreads from the initiating autoantigen to other antigens expressed in the target organ. Based on evidence from experimental models for multiple sclerosis, such "antigenic spreading" can play an important role in the exacerbation of clinical disease. We evaluated whether pathogenesis of spontaneous diabetes in NOD mice could be accelerated in a similar way when a novel autoantigen was expressed in pancreatic beta-cells. Unexpectedly, we found that the expression of the lymphocytic choriomeningitis virus nucleoprotein only led to marginal enhancement of diabetes, although such NOD-nucleoprotein mice were not tolerant to nucleoprotein. Although the frequency of nucleoprotein-specific CD8 T-cells in the pancreatic draining lymph node was comparable with the frequency of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific T-cells, more IGRP-specific CD8 T-cells were found both systemically and in the islets where there was a fourfold increase. Interestingly, and in contrast to nucleoprotein-specific CD8 T-cells, IGRP-specific T-cells showed increased CXCR3 expression. Thus, autoreactivity toward de novo-expressed beta-cell autoantigens will not accelerate autoimmunity unless large numbers of antigen-experienced autoreactive T-cells expressing the appropriate chemokine receptors are present.     

Natural history of beta-cell function in type 1 diabetes

Despite extensive and ongoing investigations of the immune mechanisms of autoimmune diabetes in humans and animal models, there is much less information about the natural history of insulin secretion before and after the clinical presentation of type 1 diabetes and the factors that may affect its course. Studies of insulin production previously published and from the Diabetes Prevention Trial (DPT)-1 suggest that there is progressive impairment in insulin secretory responses but the reserve in response to physiological stimuli may be significant at the time of diagnosis, although maximal responses are more significantly impaired. Other factors, including insulin resistance, may play a role in the timing of clinical presentation along this continuum. The factors that predict the occurrence and rapidity of decline in beta-cell function are still largely unknown, but most studies have identified islet cell autoantibodies as predictors of future decline and age as a determinant of residual insulin production at diagnosis. Historical as well as recent clinical experience has emphasized the importance of residual insulin production for glycemic control and prevention of end-organ complications. Understanding the modifiers and predictors of beta-cell function would allow targeting immunological approaches to those individuals most likely to benefit from therapy.     

Early prophylaxis with recombinant human interleukin-11 prevents spontaneous diabetes in NOD mice

We evaluated the effects of recombinant human (rh) interleukin (IL)-11 on the development of spontaneous and cyclophosphamide-induced diabetes in female NOD mice. Prolonged treatment with rhIL-11 10 microg i.p. five consecutive times a week between the 4th and 22nd weeks of age significantly suppressed both development and cumulative incidence of type 1 diabetes. Disease protection was transient because most of the animals developed type 1 diabetes within 3 months of treatment withdrawal. In contrast, rhIL-11 failed to prevent type 1 diabetes when administered for the first time to euglycemic 18-week-old NOD mice. Most likely, this discrepancy was not due to age-dependent differences in the immunological responses of NOD mice to rhIL-11 because staphylococcus aureus enterotoxin B-induced tumor necrosis factor (TNF) and IL-12 production were equally suppressed by rhIL-11 in 12- and 25-week-old NOD mice. Relative to controls, NOD mice pretreated with rhIL-11 also showed significantly diminished blood levels of TNF, interferon-gamma, and IL-12 induced by anti-CD3 antibody and/or lipopolysaccharide. The results demonstrate that rhIL-11 has powerful anti-inflammatory effects that are capable of down-regulating early immunodiabetogenic pathways in NOD mice.     

Angiotensin II type 1 receptor blockade improves beta-cell function and glucose tolerance in a mouse model of type 2 diabetes

We identified an angiotensin-generating system in pancreatic islets and found that exogenously administered angiotensin II, after binding to its receptors (angiotensin II type 1 receptor [AT1R]), inhibits insulin release in a manner associated with decreased islet blood flow and (pro)insulin biosynthesis. The present study tested the hypothesis that there is a change in AT1R expression in the pancreatic islets of the obesity-induced type 2 diabetes model, the db/db mouse, which enables endogenous levels of angiotensin II to impair islet function. Islets from 10-week-old db/db and control mice were isolated and investigated. In addition, the AT1R antagonist losartan was administered orally to 4-week-old db/db mice for an 8-week period. We found that AT1R mRNA was upregulated markedly in db/db islets and double immunolabeling confirmed that the AT1R was localized to beta-cells. Losartan selectively improved glucose-induced insulin release and (pro)insulin biosynthesis in db/db islets. Oral losartan treatment delayed the onset of diabetes, and reduced hyperglycemia and glucose intolerance in db/db mice, but did not affect the insulin sensitivity of peripheral tissues. The present findings indicate that AT1R antagonism improves beta-cell function and glucose tolerance in young type 2 diabetic mice. Whether islet AT1R activation plays a role in the pathogenesis of human type 2 diabetes remains to be determined.     

Gene expression profiles define a key checkpoint for type 1 diabetes in NOD mice

cDNA microarrays with >11,000 cDNA clones from an NOD spleen cDNA library were used to identify temporal gene expression changes in NOD mice (1-10 weeks), which spontaneously develop type 1 diabetes, and changes between NOD and NOD congenic mice (NOD.Idd3/Idd10 and NOD.B10Sn-H2(b)), which have near zero incidence of insulitis and diabetes. The expression profiles identified two distinct groups of mice corresponding to an immature (1-4 weeks) and mature (6-10 weeks) state. The rapid switch of gene expression occurring around 5 weeks of age defines a key immunological checkpoint. Sixty-two known genes are upregulated, and 18 are downregulated at this checkpoint in the NOD. The expression profiles are consistent with increased antibody production, antigen presentation, and cell proliferation associated with an active autoimmune response. Seven of these genes map to confirmed diabetes susceptibility regions. Of these seven, three are excellent candidate genes not previously implicated in type 1 diabetes. Ten genes are differentially expressed between the NOD and congenic NOD at the immature stage (Hspa8, Hif1a, and several involved in cellular functions), while the other 70 genes exhibit expression differences during the mature (6-10 week) stage, suggesting that the expression differences of a small number of genes before onset of insulitis determine the disease progression.