Autoimmune Diabetes In Mice Research Articles

Intramuscular transfer of naked calcitonin gene-related peptide gene prevents autoimmune diabetes induced by multiple low-dose streptozotocin in C57BL mice.

The imbalance of Th1/Th2 subsets is an important pathogenic mechanism for insulin-dependent diabetes mellitus (IDDM). Calcitonin gene-related peptide (CGRP) has been found to play important roles in the regulation of T lymphocytes. We hypothesize that exogenous CGRP administration during insulitis may modulate the balance of Th lymphocytes, thereby providing a therapeutic intervention for IDDM. We established CGRP gene transfer by naked plasmid injection into the skeletal muscles with electroporation enhancement. The effect of CGRP gene transfer on pathogenesis of IDDM was observedin autoimmune diabetic C57BL mice induced by multiple low-dose streptozotocin (MLDS) administration. The treatment significantly decreased morbidity of diabetes, ameliorated hyperglycemia and insulin deficiency, and inhibited lymphocyte infiltration into the islets, indicating the protection of beta cells against autoimmune destruction. CGRP gene transfer significantly inhibited T cell proliferation and secretion of the Th1 cytokine IFN-gamma, increased the level of the Th2 cytokine IL-10, but had no effect on IL-4 and TGF-beta1 secretion. CGRP gene transfer also decreased IL-12 and IFN-gamma levels in peritoneal effusion. Our results demonstrate that CGRP gene transfer selectively suppresses the pro-inflammatory Th1 subsets and promote anti-inflammatory Th2 subsets, resulting in amelioration of beta cell destruction and reduction of IDDM occurrence in mice with MLDS-induced diabetes.

Activation of mouse macrophage by soluble endogenous murine leukemia virus (MuLV) envelope protein

We identified recently an endogenous murine leukemia virus (MuLV) envelope protein as a new autoantigen reactive with autoimmune diabetic mouse sera and observed immunosuppressive activity of this envelope protein. In the present study, to elucidate the mechanism involved, we treated macrophages with the envelope protein and investigated activation of macrophage. We found enhancements of iNOS mRNA and nitrite in envelope protein-treated RAW264.7 cells and peritoneal macrophages. The stimulation was highly envelope protein-specific, and also time- and dose-dependent. The activation pattern was similar to that elicited by lipopolysaccharide (LPS) since the envelope protein showed a synergistic effect on macrophage activation in conjunction with interferon gamma (IFN-γ). Furthermore, deacylated LPS as a competitive inhibitor of LPS showed inhibition of envelope protein-mediated macrophage activation. These data show that MuLV envelope protein can be a new macrophage activator and suggest that the retroviral envelope protein may elicit immunosuppressive activity through macrophage activation.

Disease resistant, NOD-related strains reveal checkpoints of immunoregulation in the pancreas

The autoimmune diabetic NOD mouse serves as a model for human type 1 diabetes. Disease development is due to islet beta cell destruction in the context of immune cell infiltration of islets and inflammatory changes throughout the pancreas. In the present study we tried to identify immune reactivity patterns in the pancreas associated with diabetes resistance in NOD-related mouse strains. The pancreata of diabetes-prone female NOD/LtJ, NOD/Bom and of genetically related but diabetes-resistant strains; NOR, NON, NON.NOD-H2g7, NOD.NON-H-2nbl were obtained at the age of 70 days for semiquantitative analysis of insulitis and of mRNA expression by reverse transcriptase PCR. In addition, the response to a single dose of cyclophosphamide for synchronizing and accelerating the progression of insulitis was determined. The progression of insulitis and immune gene expression in response to cyclophosphamide revealed characteristic differences between the six strains. NOD/LtJ and NOD/Bom mice were found significantly to upregulate pancreatic IL-12p40 and IL-18 expression after cyclophosphamide treatment, followed by an increase in IFN-gamma mRNA levels. In contrast, the two MHC-haplotype H-2nbl expressing strains either up-regulated neither IL-12/IL-18 nor IFN-gamma gene expression. The two strains sharing MHC haplotype H-2g7 expression with NOD did respond to cyclophosphamide with IL-12p40/IL-18 gene expression. However, NON.NOD-H-2g7 mice failed to progress to IFN-gamma gene expression. NOR mice progressed to IFN-gamma expression but exhibited sustained IL-4 gene expression. Only severe intra-insulitis was associated with the expression of inducible NO synthase. The comparison of diabetes-prone and diabetes-resistant strains revealed three checkpoints of immune regulation in the pancreas. The earliest checkpoint is the induction of an IL-12p40/IL-18 response in innate immune or antigen-presenting cells. The next level of control is at the induction of IFN-gamma gene expression, and a third checkpoint is the maintenance or loss of antagonistic Th2 type reactions.

Implications of altered apoptosis in diabetes mellitus and autoimmune disease.

Lymphocyte development, selection and education represent tightly controlled immune processes that normally prevent autoimmunity. Lymphocyte development requires cellular selection through apoptosis to remove potentially autoreactive cells. Dysregulated apoptosis, both interrupted as well as accetuated apoptosis, are now demonstrated as central defects in diverse human and murine autoimmune disease. In murine models of autoimmune lupus, mutations in cell death receptor CD95 (Fas) and its ligand CD95L (FasL) have been identified; these errors create a lymphoid system resistant to apoptosis. In contract, select lymphoid subpopulations of auto immune diabetic mice have accelerated apoptosis due to faulty activation of transcription factor NF-kappaB that normally protects against apoptotic death. The genetic basis of interrupted NF-kappaB in diabetes is a gene defect in an essential subunit of the proteasome. Although no specific gene in most common forms of human autoimmune disease has been identified, functional assays repeatedly demonstrate apoptotic defects in multiple cellular signaling pathways for cell death.

Remission in models of type 1 diabetes by gene therapy using a single-chain insulin analogue.

A cure for diabetes has long been sought using several different approaches, including islet transplantation, regeneration of beta cells and insulin gene therapy. However, permanent remission of type 1 diabetes has not yet been satisfactorily achieved. The development of type 1 diabetes results from the almost total destruction of insulin-producing pancreatic beta cells by autoimmune responses specific to beta cells. Standard insulin therapy may not maintain blood glucose concentrations within the relatively narrow range that occurs in the presence of normal pancreatic beta cells. We used a recombinant adeno-associated virus (rAAV) that expresses a single-chain insulin analogue (SIA), which possesses biologically active insulin activity without enzymatic conversion, under the control of hepatocyte-specific L-type pyruvate kinase (LPK) promoter, which regulates SIA expression in response to blood glucose levels. Here we show that SIA produced from the gene construct rAAV-LPK-SIA caused remission of diabetes in streptozotocin-induced diabetic rats and autoimmune diabetic mice for a prolonged time without any apparent side effects. This new SIA gene therapy may have potential therapeutic value for the cure of autoimmune diabetes in humans.

Suppression of insulitis and diabetes in B cell-deficient mice treated with streptozocin: B cells are essential for the TCR clonotype spreading of islet-infiltrating T cells.

In order to clarify the role of B cells in the development of insulitis and diabetes, B cell-deficient (B(-)) mice treated with streptozocin (STZ) were studied. The extent of insulitis and the cumulative incidence of diabetes were significantly suppressed in B(-) mice (P < 0.0001), indicating that B cells are crucial for the progression of insulitis and diabetes. Accumulation of both CD4(+) T cells and B cells was observed in islets of B(+) mice, while CD4(+) T cells but not B cells were found in B(-) mice. A few CD8(+) T cells and macrophages were detectable in both types of mice. The immunohistochemical study did not reveal any change in the subpopulations of infiltrating lymphocytes except for the absence of B cells in the B(-) mice. TCR V(beta) gene repertoire usage of islet-infiltrating T cells was restricted to some extent in the B(+) or B(-) mice, but there was no significant difference between the B(+) and B(-) mice, suggesting that the initial islet-reactive T cell response can occur in the absence of B cells. In contrast, TCR clonotype spreading of islet-infiltrating T cells was significantly suppressed in B(-) mice compared with B(+) mice (P < 0.0001). These data suggest that initial priming of T cells is not impaired and TCR V(beta) repertoire usage is not limited by the lack of B cells, while B cells are important essentially for the spreading of islet-infiltrating clonal T cells in autoimmune diabetic mice induced with STZ.

Inhibition of the Cytokine-Mediated Inducible Nitric Oxide Synthase Expression in Rat Insulinoma Cells by Phenyl N-tert-Butylnitrone

Cytokines and nitric oxide (NO) have been implicated in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). We have shown that the spin-trapping agent phenyl N-tert-butylnitrone (PBN) protects against streptozotocin (STZ)-induced IDDM in mice. In order to gain more insights into the mechanism(s) of the protective action of PBN against IDDM, we have investigated the effect of this compound on the cytokine-induced NO generation (measured as nitrite) in rat insulinoma RIN-5F cells. Our results demonstrate that PBN cotreatment prevents the generation of nitrite by RIN-5F cells induced by treatment with tumor necrosis factor-α, interleukin 1β, and interferon-γ in a dose-dependent fashion. The generation of NO as a result of cytokine treatment and the inhibitory effect of PBN were further confirmed by electron paramagnetic resonance spectroscopy. Aminoguanidine, a selective inhibitor of inducible nitric oxide synthase (iNOS), abolished the cytokine-induced nitrite generation whereas N-nitro-l-arginine, an inhibitor more selective for other NOS isoforms, was significantly less effective. Western and Northern analyses demonstrated that PBN inhibits the cytokine-mediated expression of iNOS at the transcriptional level. Cytokine-induced nitrite formation was also inhibited by the two antioxidant agents α-lipoic acid and N-acetylcysteine. These results indicate that PBN protects against IDDM at least in part by prevention of cytokine-induced NO generation by pancreatic β-cells.

Pioglitazone prevents mice from multiple low-dose streptozotocin-induced insulitis and diabetes

Macrophage infiltration into pancreatic islets is thought to be an initial event inducing insulitis in the development of type 1 diabetes. Thiazolidinedione is a direct ligand for peroxisome proliferator-activated receptor-γ, recently reported to inhibit macrophage activation, including cytokine production and type 2 nitric oxide synthase expression. We investigated the effect of pioglitazone, a thiazolidinedione compound, on the development of multiple low-dose streptozotocin (MLDS)-induced autoimmune diabetes in mice. CD-1 mice intraperitoneally injected with five daily sub-diabetogenic doses (30 or 40 mg/kg body weight) of streptozotocin developed mononuclear cell infiltration in and around islets, followed by hyperglycemia. Oral administration of pioglitazone (0.01% food admixture) from 7 days before the first streptozotocin injection prevented or delayed the development of diabetes induced by MLDS. Histologically, pioglitazone blocked the infiltration of mononuclear cells into islets in MLDS mice. Peritoneal macrophages from MLDS mice at day-7 produced significantly large amount of nitric oxide compared with those from control mice. Such activation of peritoneal macrophages was not observed in pioglitazone-treated MLDS mice. These findings suggest that pioglitazone blocks the autoimmune process in the development of MLDS diabetes, partly by inhibiting the macrophage activation.

PRIMARY NONFUNCTION OF ISLET XENOGRAFTS IN SPONTANEOUSLY DIABETIC AUTOIMMUNE NOD MICE: CORRELATION WITH ELEVATED NON-T CELL CYTOKINES AND FASL EXPRESSION IN THE GRAFTS.

522 Primary nonfunction (PNF) and early graft destruction are obstacles for the application of islet xenotransplantation in autoimmune type 1 diabetes. The first aim of this study was to investigate the role of autoimmune disease recurrence in PNF and xenograft survival in autoimmune NOD mice after PVG (c+) rat islet transplantation. The second aim was to examine the mediators of this early islet destruction. Results: Spontaneously diabetic autoimmune NOD mice had a shorter islet xenograft survival (5.4 ± 2.3 days) compared to chemically diabetic C57Bl/6 or old NOD mice (11.3 ± 1.2 days, p<0.01 and 9.7 ± 3.7 days, p<0.05). Also a higher incidence in PNF (44%) was noted in these autoimmune diabetic mice compared to non-autoimmune diabetic C57B1/6 or old NOD mice (10% and 29%). Analysis of cytokine mRNA levels locally in the xenografts showed clearly higher IL-1α, TNF-α and IL-6 levels in spontaneously diabetic autoimmune NOD mice 8 hours posttransplantation, rapidly decreasing after 16 hours. This early rise in macrophage-derived cytokines was mirrored by high mRNA levels of Fas ligand (Fas-L), a mediator of cell death in Fas expressing cells such as β cells. In the islet xenografts of the non-autoimmune diabetic mice (chemically diabetic C57Bl/6 or old NOD mice), elevated levels of IL-1α, TNF-α and IL-6 were only noted at 16 hours after transplantation. These high macrophage cytokine levels were now however not paralled by a rise in Fas-L mRNA. Another difference between autoimmune and non-autoimmune mice were the levels of the suppressive cytokine, TGF-β, being clearly higher (2 fold) in the non-autoimmune situation. T-cell cytokines (IL-2, IL-4, IL-12 and INF-γ) in islet xenografts of both autoimmune and non-autoimmune diabetic mice were absent or similar with background levels in the first 24 hours after transplantation, as were T cells on immunohistochemistry. In conclusion, these data suggest a role for the autoimmune memory in the occurrence of PNF and islet xenograft destruction in the spontaneously diabetic autoimmune NOD mice. The destruction of islets seems however to be mediated primarily by non-T cell types such as macrophages.

Fas on renal parenchymal cells does not promote autoimmune nephritis in MRL mice

Although Fas on pancreatic islets promotes autoimmune diabetes in mice, the role of Fas expression on kidney parenchymal cells during autoimmune disease is unknown. To determine whether Fas on renal parenchymal cells promotes autoimmune renal destruction, we compared apoptosis and pathology in Fas-intact and Fas-deficient kidneys in an autoimmune milieu. For this purpose, we transplanted single, normal kidneys from MRL-++ (Fas-intact) mice (3 months of age) into age-matched, congenic MRL-Faslpr (Fas-deficient) recipients after removal of nephritic kidneys. These Fas-intact kidneys were compared with Fas-deficient nephritic kidneys. There is a progressive increase of FasL on kidney-infiltrating cells and Fas and FasL on renal parenchymal cells in MRL-++ kidneys during engraftment (0, 2, 4-6, and 8 weeks). By comparison, we detected an increase in FasL in MRL-Faslpr kidneys (3 to 5 months of age), whereas Fas was not detectable. The engagement of T cells bearing FasL with Fas expressing tubular epithelial cells (TECs) induced TEC apoptosis in vitro. However, apoptosis and pathology were similar in kidneys (MRL-++, 8 weeks postengraftment vs. MRL-Faslpr, 5 months) with equivalent amounts of FasL-infiltrating cells or FasL TECs, regardless of Fas on renal parenchymal cells. The expression of Fas on renal parenchymal cells does not increase apoptosis or promote renal disease in MRL-++ mice. We speculate that the autoimmune milieu evokes mechanisms that mask, counter, or pre-empt Fas-FasL-initiated apoptosis in MRL kidneys.

Prevention of experimental autoimmune diabetes in mice by treatment with leflunomide

Prevention of experimental autoimmune diabetes in mice by treatment with leflunomide

Prolongation of porcine islet xenograft survival in mice after therapy with immunosuppressive peptides.

Recently, peptides derived from the heavy chain of HLA-B2702 have been shown to modulate immune responses. In this study, we examined the use of these peptides for immunosuppression in a pig to mouse islet xenograft model. Purified porcine islets were transplanted in autoimmune (non-obese diabetic) and non-autoimmune (streptozotocin-injected CBA or C57/Bl6) diabetic mice. Various dosing regimens of HLA-derived peptides with and without antilymphocyte therapy were administered to recipient mice. Graft rejection was determined by daily serum glucose determinations, and, at selected time points, grafts were removed to demonstrate function and provide immunohistochemical examination. HLA-derived peptides were demonstrated to prolong graft survival in both pretransplant and posttransplant treatment regimens. This effect was increased with concomitant antilymphocyte therapy. Further elucidation of the mechanism of action of these immunomodulatory peptides may help in the development of novel immunosuppressive protocols.

ISLET-SPLENOCYTE CO-TRANSPLANTATION ABOLISHES AUTOIMMUNE-DRIVEN PRIMARY NON-FUNCTION IN THE NON-OBESE DIABETIC MOUSE

158 Clinical islet transplantation remains relatively unsuccessful in comparison to whole pancreas transplantation. Evidence from the BB rat model of spontaneous diabetes demonstrates that isolated islets transplants are peculiarly sensitive to recurrent autoimmunity but co-transplantation of pancreatic lymph node cells from diabetes resistant donors leads to engraftment of a donor T cell population (RT6+) that prevents recurrent autoimmune destruction of islet isografts and allografts. This model is limited by the severe T lymphopenia of the BB rat. Demonstration of this finding in a non-lymphopenic model, the non-obese diabetic (NOD) mouse, would significantly increase the likelihood that this finding is clinically applicable. METHODS: 500 islets from 4-6 week old NOD mouse donors were transplanted under the renal capsule of autoimmune diabetic (NOD-Au) or streptozocin (300 mg/kg) diabetic (NOD-Sz) mice. Some cohorts simultaneously received ˜75 million H-2 matched non-obese resistant (NOR) splenocytes (SPL) by tail vein and/or recipient pre-treatment with the CD4 depleting mab GK1.5 400 µg/kg on days -7,-2,-1 and 0. FACS analysis confirmed CD4 depletion to < 1%. Graft failure was determined by blood glucose >200 mg/dl on two consecutive days. Immunoblot analysis assessed mouse RT6 expression in prediabetic and diabetic NOD and NOD islet/SPL recipients. RESULTS: Table Immunoblot analysis revealed that splenocyte RT6 expression was identical in naive prediabetic NOD, NOR and NOD recipients of successful islet transplants but not autoimmune diabetic NOD animals. CONCLUSIONS: These results demonstrate that primary nonfunction of islet transplants in autoimmune diabetic NOD recipients is autoimmune-driven since islet transplants in chemically diabetic NOD mice function indefinitely. CD4 depletion and NOR splenocyte co-transplantation restores RT6 expression to pre-diabetic levels leading to ablation of the autoimmune-driven primary non-function of islet transplants. This data confirms that in a non-lymphopenic animal model recurrent autoimmunity can be avoided with addition of donor immune cells at the time of transplantation.Table

Immunosuppression preventing concordant xenogeneic islet graft rejection is not sufficient to prevent recurrence of autoimmune diabetes in nonobese diabetic mice.

We and others have reported previously that the immunosuppressant, leflunomide (Lef), can prevent allogeneic and xenogeneic islet graft rejection in streptozocin (STZ)-induced diabetic animals. However, whether Lef required to prevent islet graft rejection is sufficient to prevent the recurrence of autoimmune diabetes has not been addressed. The effect of Lef on concordant xenogeneic islet graft in STZ-induced diabetic mice and autoimmune nonobese diabetic (NOD) mice were studied. Then, whether Lef prevents the onset of spontaneous diabetes in young NOD mice and the recurrence of diabetes after major histocompatibility complex (MHC)-matched islet transplantation in diabetic NOD mice were investigated. In STZ-induced diabetic BALB/c mice, Lef treatment significantly prolonged rat islet graft survival. However, Lef could not significantly prolong rat islet graft survival in autoimmune diabetic NOD mice. For prevention studies, treatment with Lef at 30 mg/ kg/day from 4 weeks to 20 weeks of age significantly reduced the incidence of spontaneous diabetes in NOD mice. However, when the NOD mice were treated from 8 to 24 weeks of age, the incidence of spontaneous diabetes was not significantly reduced as compared to the incidence of diabetes in the untreated female NOD mice at 28 weeks of age. Finally, in the MHC-matched islet transplant model, Lef could not significantly prolong MHC-matched nonobese diabetes-resistant mice islet graft survival in NOD mice. Lef preventing concordant xenogeneic islet graft rejection is not sufficient to prevent the recurrence of autoimmune diabetes in NOD mice. We believe that controlling autoimmunity after islet transplantation will lead the way to promote successful clinical islet transplantation in the future.

Cloned T Cells from a Recent Onset IDDM Patient Reactive with Insulin B-chain

Insulin-dependent diabetes mellitus (IDDM) results from selective auto-immune destruction of insulin producing β-cells. T-cell reactivity and auto-antibodies to several islet proteins such as insulin, GAD and IA-2 are associated with IDDM in mice and men. In NOD mice, the majority of T cells from insulitis specifically recognize the insulin B-chain peptide amino acid 9-22, in contrast to the periphery where the precursor frequency is much lower. It is important to note that these cells are diabetogenic. Surprisingly, the same insulin B-chain region contains epitopes recognized by protective T cells. In fact, autoimmune diabetes in NOD mice could be prevented by prophylactic treatment with this immunodominant T-cell epitope. In humans, however, no immunodominant regions of insulin have yet been defined. We have isolated and characterized a human insulin-specific T-cell clone that was derived from peripheral blood of a newly diagnosed IDDM patient. This patient displayed weakly positive primary T-cell responses to insulin. The peptide recognized by the clone was mapped to the insulin B chain (B:11-27). Functionally, the human insulin-specific CD4+T cells displayed a Th1/0 like cytokine profile and were restricted by HLA-DR. The previously proposed alternative superantigen-like binding of insulin-B chain peptide outside of the peptide binding groove of HLA-DR could not be confirmed, since T-cell recognition was inhibited in competition experiments of insulin-B chain peptide with HLA-DR16 binding influenza peptide HA307-319. Our results indicate that human clonal T cells isolated from a recent onset IDDM patient recognize an epitope overlapping with the insulin B-chain region that is immunodominant and potentially therapeutic in NOD mice. This observation may be useful in studying the role of insulin-specific T cells in IDDM, and may eventually help to establish peptide-based immunotherapies in IDDM.

Effect of leflunomide and cyclosporine on concordant xenogeneic islet transplantation in streptozotocin-induced and autoimmune diabetic mice

Effect of leflunomide and cyclosporine on concordant xenogeneic islet transplantation in streptozotocin-induced and autoimmune diabetic mice

Inducible nitric oxide synthase (iNOS) in pancreatic islets of nonobese diabetic mice: identification of iNOS- expressing cells and relationships to cytokines expressed in the islets

Inflammatory cytokines and nitric oxide (NO) are candidate mediators of pancreatic islet beta-cell destruction in insulin-dependent diabetes mellitus. In this study, we used a semiquantitative PCR assay to measure levels of messenger RNA (mRNA) expression of the inflammatory cytokines, interleukin-1 alpha (IL-1 alpha), tumor necrosis factor-alpha, and interferon-gamma (IFN gamma), and of the inducible form of NO synthase (iNOS) in mononuclear leukocytes isolated from pancreatic islets of autoimmune diabetes-prone nonobese diabetic (NOD) female mice. We found that mRNA levels of iNOS, IL-1 alpha, and IFN gamma in islet mononuclear leukocytes increased from 5 weeks of age to onset of diabetes ( > 13 weeks of age). To determine whether increased iNOS, IL-1 alpha, and IFN gamma mRNA expressions were related to diabetes development, we compared mRNA levels of these molecules in mononuclear leukocytes from islets of 12 week-old diabetes-prone NOD female mice and three groups of 12-week-old mice with low diabetes risk: NOD female mice injected with complete Freund's adjuvant at 4 weeks of age, NOD male mice, and BALB/c female mice that do not develop diabetes. We found that iNOS, IL-1 alpha, and IFN gamma mRNA levels were higher in mononuclear leukocytes from islets of diabetes-prone NOD female mice than in those from mice correlated with IL-1 alpha and IFN gamma mRNA levels. By using specific antibodies and immunohistochemical methods, we localized iNOS in macrophages as well as in beta-cells of islets from diabetes-prone NOD female mice. These findings suggest that IL-1 alpha and IFN gamma may promote islet beta-cell destruction at least in part by up-regulating iNOS expression an No production by both macrophages and beta-cells in the islets of autoimmune diabetes-prone NOD mice.

Inducible nitric oxide synthase (iNOS) in pancreatic islets of nonobese diabetic mice: identification of iNOS- expressing cells and relationships to cytokines expressed in the islets.

Inflammatory cytokines and nitric oxide (NO) are candidate mediators of pancreatic islet beta-cell destruction in insulin-dependent diabetes mellitus. In this study, we used a semiquantitative PCR assay to measure levels of messenger RNA (mRNA) expression of the inflammatory cytokines, interleukin-1 alpha (IL-1 alpha), tumor necrosis factor-alpha, and interferon-gamma (IFN gamma), and of the inducible form of NO synthase (iNOS) in mononuclear leukocytes isolated from pancreatic islets of autoimmune diabetes-prone nonobese diabetic (NOD) female mice. We found that mRNA levels of iNOS, IL-1 alpha, and IFN gamma in islet mononuclear leukocytes increased from 5 weeks of age to onset of diabetes ( > 13 weeks of age). To determine whether increased iNOS, IL-1 alpha, and IFN gamma mRNA expressions were related to diabetes development, we compared mRNA levels of these molecules in mononuclear leukocytes from islets of 12 week-old diabetes-prone NOD female mice and three groups of 12-week-old mice with low diabetes risk: NOD female mice injected with complete Freund's adjuvant at 4 weeks of age, NOD male mice, and BALB/c female mice that do not develop diabetes. We found that iNOS, IL-1 alpha, and IFN gamma mRNA levels were higher in mononuclear leukocytes from islets of diabetes-prone NOD female mice than in those from mice correlated with IL-1 alpha and IFN gamma mRNA levels. By using specific antibodies and immunohistochemical methods, we localized iNOS in macrophages as well as in beta-cells of islets from diabetes-prone NOD female mice. These findings suggest that IL-1 alpha and IFN gamma may promote islet beta-cell destruction at least in part by up-regulating iNOS expression an No production by both macrophages and beta-cells in the islets of autoimmune diabetes-prone NOD mice.

Pathogenic and protective roles of CD45RB(low) CD4+ cells correlate with cytokine profiles in the spontaneously autoimmune diabetic mouse.

The adoptive transfer of splenocytes from diabetic NOD mice to NOD-scid/scid (NOD-scid) recipients results in diabetes. This model was used to test the effect of cotransfer of splenocyte subsets from young nondiabetic NOD mice. As shown previously in other NOD models, the CD4+ subset from young nondiabetic mice significantly delayed the onset of diabetes in splenocyte cotransfers (P < 0.001). The data presented here showed that the development of diabetes in NOD-scid recipients correlated with a rapid increase in peripheral CD45RB(low) CD4+ cells. However, the CD45RB(low) subset of CD4+ cells from young nondiabetic mice protected from diabetes transfer in this model. We therefore examined whether CD45RB(low) CD4+ cells from diabetic mice were pathogenic rather than protective. CD45RB(low) CD4+ splenocytes from diabetic NOD mice were transferred along with CD8+ splenocytes from diabetic mice into NOD-scid recipients, and all of the recipients became diabetic within 5 weeks posttransfer. In contrast, no recipients (0 of 10) of CD45RB(high) CD4+ cells along with CD8+ splenocytes from diabetic mice became diabetic within 5 weeks posttransfer (P < 0.001). A correlate for the difference between CD45RB(low) CD4+ cells from diabetic NOD mice and CD45RB(low) CD4+ cells from nondiabetic mice, which showed protective effect in splenocyte cotransfers, was found in cytokine production after stimulation with anti-CD3 antibodies in vitro. CD45RB(low) CD4+ cells from diabetic mice showed a significantly higher ratio (approximately fivefold) of gamma-interferon (IFN-gamma) to interleukin (IL)-4 when compared with CD45RB(low) CD4+ cells from nondiabetic mice (P < 0.001). In conclusion, the function of the CD45RB(low) population of CD4+ cells changes from a protective to a pathogenic one during the development of disease in the NOD mouse. This change in function correlates with cytokine production in vitro; increased IFN-gamma-to-IL-4 ratio is associated with pathogenic potential and occurs coincident with (or after) the onset of diabetes.

Pathogenic and protective roles of CD45RB(low) CD4+ cells correlate with cytokine profiles in the spontaneously autoimmune diabetic mouse

Pathogenic and protective roles of CD45RB(low) CD4+ cells correlate with cytokine profiles in the spontaneously autoimmune diabetic mouse