Studies of concanavalin A in nonobese diabetic mice. II. Lymphocyte tracking and phenotype responses

We studied the tracking of 51chromium-labeled (resting) and Con A-induced (activated) tritiated thymidine-labeled syngeneic lymphocytes to adrenal, blood, brain, heart, liver, lymph nodes, pancreas, spleen, testis/ovary, thymus and thyroid in prediabetic, nonobese diabetic (NOD) mice and in age- and sex-matched C57BL/6 mice. 51Chromium-labeled cells showed no significant difference between strains in tracking to any tissue except lymph nodes (decreased in NOD vs. C57, P less than .05). Con A incubation resulted in no difference between strains in lymphocyte tracking to lymph nodes, but NOD mice had increased pancreatic tracking with Con A-incubated cells compared to C57 mice (P less than .05). Female NOD mice had reduced thymic tracking (P = .001) compared to C57 controls. Positive selection experiments showed the Con A-responsive cell to be a T cell. Both Lyt2+ (CD8+) and L3T4+ (CD4+) enriched T cell populations showed a labeling response to Con A. After 48 h of Con A incubation, the L3T4+/Lyt2+ ratio increased in splenocytes from NOD mice (P less than .05), whereas it decreased in C57 controls (P less than .01). Over the course of 6 days in culture, NOD splenocytes exposed to Con A characteristically exhibited a delayed expansion of the Lyt2+ population. We conclude that Con A incubation of NOD splenocytes results in T cells that, when reinfused, avoid the thymus and track preferentially to the pancreas. Con A immunomodulation as potential treatment for autoimmune disease warrants further study in this murine model.     

Prevention of diabetes in nonobese diabetic mice by dendritic cell transfer.

The purpose of this study was to determine the effect of dendritic cell (DC) transfers on the incidence of diabetes in female nonobese diabetic (NOD) mice. Groups of 4-wk-old NOD female mice were given a single foot pad of DCs (70-90% purity) isolated from the draining lymph nodes (LN) of the pancreas (PLN), the cervical LNs, or the axillary/inguinal LNs. In addition, other groups of NOD mice received purified spleen DCs, purified PLN T cells (the major contaminating population in DC preparations), or the injection vehicle PBS. All groups were monitored for diabetes for one year. Significant protection from diabetes was observed in NOD mice receiving greater than 1 x 10(4) PLN DCs in comparison to mice receiving other DCs populations, PLN T cells, or PBS (P less than 0.05). The pancreata of NOD mice that received PLN DCs demonstrated significantly lower levels of lymphocytic infiltration in the islets that age-sex matched nondiabetic female NOD control mice (P less than 0.05). LN cells from nondiabetic NOD mice that received PLN DC protected irradiated female recipients from the adoptive transfer of diabetes to a greater degree than LN cells from age and sex matched nondiabetic female NOD mice that did not receive PLN DC transfers at 36 d (P = 0.014) and at 1 yr (P = 0.0015) after transfer. These data suggest that the PLN DC transfers are able to modulate autoimmunity and limit diabetes expression in the NOD mouse. PLN DCs transfers may regulate autoimmunity by the induction of regulatory cells.     

Androgen treatment prevents diabetes in nonobese diabetic mice

The nonobese diabetic (NOD) mouse strain provides a model system for human autoimmune diabetes. This disease model is extensively used not only to examine the etiology and pathogenesis of diabetes, but also as a means to evaluate therapies. In NOD mice, the disease progresses from insulitis to islet destruction and clinical diabetes in a high percentage of female mice. In this study, androgen therapy, begun after the onset of insulitis, was found to prevent islet destruction and diabetes without eliminating the islet inflammation in female NOD mice. However, diabetes can be adoptively transferred into such hormone-treated recipients. The prevention of disease onset by androgen is likely due to the hormonal alteration of the development or function of the immune cells necessary for islet destruction.     

Reversal of beta-cell suppression in vitro in pancreatic islets isolated from nonobese diabetic mice during the phase preceding insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is characterized by a progressive autoimmune destruction of the pancreatic beta-cells. One of the best-suited animal models for IDDM is the nonobese diabetic (NOD) mouse. In this investigation pancreatic islets were isolated from female NOD mice aged 5-7, 8-11, and 12-13 wk and examined immediately (day 0) or after 7 d of culture (day 7). The mice showed a progressive disturbance in glucose tolerance with age, and a correspondingly increased frequency of pancreatic insulitis. Islets isolated from the oldest mice often contained inflammatory cells on day 0, which resulted in an elevated islet DNA content. During culture these islets became depleted of infiltrating cells and the DNA content of the islets decreased on day 7. Islets of the eldest mice failed to respond with insulin secretion to high glucose, whereas a response was observed in the other groups. After culture all groups of islets showed a markedly improved insulin secretion. Islets from the 12-13-wk-old mice displayed a lower glucose oxidation rate at 16.7 mM glucose on day 0 compared with day 7. Islet (pro)insulin and total protein biosynthesis was essentially unaffected. In conclusion, islets obtained from 12-13-wk-old NOD mice exhibit an impaired glucose metabolism, which may explain the suppressed insulin secretion observed immediately after isolation. This inhibition of beta-cell function can be reversed in vitro. Thus, there may be a stage during development of IDDM when beta-cell destruction can be counteracted and beta-cell function restored, provided the immune aggression is arrested.     

Viruses as therapeutic agents. I. Treatment of nonobese insulin- dependent diabetes mice with virus prevents insulin-dependent diabetes mellitus while maintaining general immune competence

A situation in which virus can be used as a therapeutic agent to prevent a lethal autoimmune disease is explored. Nonobese insulin-dependent diabetes (NOD) mice spontaneously develop insulin-dependent diabetes mellitus (IDDM), characterized by lymphocytic infiltration into the islets of Langerhans and beta cell destruction, resulting in hypoinsulinemia, hyperglycemia, ketoacidosis, and death. Infection of NOD mice with lymphocytic choriomeningitis virus (LCMV) aborts the autoimmune manifestations and resultant IDDM. The viruses' effect is on a subset of CD4+ lymphocytes. Ablating this autoimmune diabetes does not significantly alter immune responses to a variety of non-LCMV antigens that require CD4+ lymphocyte participation. The prevention of IDDM associated with viral therapy is maintained throughout the life spans of NOD mice.     

Recombinant human tumor necrosis factor alpha suppresses autoimmune diabetes in nonobese diabetic mice.

We previously reported that administration of a streptococcal preparation (OK-432) inhibited insulitis and development of autoimmune diabetes in nonobese diabetic (NOD) mice and BB rats as animals models of insulin-dependent diabetes mellitus. In this study, we screened various cytokines that could be induced by OK-432 in vivo, for their preventive effect against diabetes in NOD mice. Among recombinant mouse IFN gamma, human IL1 alpha, human IL2, mouse granulocyte-macrophage colony-stimulating factor and human TNF alpha, only human TNF alpha suppressed insulitis and significantly (P less than 0.001) inhibited development of diabetes. NOD mice were the lowest producers of the mRNA of TNF and serum TNF on stimulation with OK-432 or with IFN gamma plus LPS, compared with C57BL/6, C3H/He, and Balb/c mice. The results imply a role for low productivity of TNF in the pathogenesis of autoimmune diabetes in NOD mice.     

A critical role for lymphotoxin-beta receptor in the development of diabetes in nonobese diabetic mice

To assess the role of lymphotoxin-beta receptor (LTbetaR) in diabetes pathogenesis, we expressed an LTbetaR-Fc fusion protein in nonobese diabetic (NOD) mice. The fusion protein was expressed in the embryo, reached high levels for the first 2 wk after birth, and then declined progressively with age. High expression of LTbetaR-Fc blocked diabetes development but not insulitis. After the decline in chimeric protein concentration, mice became diabetic with kinetics similar to the controls. Early expression of fusion protein resulted in disrupted splenic architecture. However, primary follicles and follicular dendritic cells, but not marginal zones, developed in aged mice. Hence, LTbetaR signaling is required for diabetes development and regulates follicular and marginal zone structures via qualitatively or quantitatively distinct mechanisms.     

Breaking self-tolerance in nonobese diabetic mice

Unresponsiveness to self is maintained through two mechanisms of immune regulation: thymic-negative selection and peripheral tolerance. Although thymic-negative selection is a major mechanism to eliminate self-reactive T cells, normal mice have readily detectable populations of T cells reactive to self-proteins but do not exhibit autoimmune responses. It has been postulated that autoimmune disease results from breakdown or loss of peripheral tolerance. We present data that demonstrate that peripheral tolerance or unresponsiveness to self can be broken in nonobese diabetic (NOD) mice. Immunization of NOD mice (but not of conventional mice) with self-peptides caused an immune response to self-peptide with resultant autoproliferation of peripheral lymphocytes. Autoproliferation of self-reactive T cells in NOD mice resulted from the recognition and proliferation of the activated T cells to endogenously processed and presented self-antigens. This loss of self-tolerance demonstrated in vitro may well be the basis of NOD autoimmune disease in vivo.     

A defect in interleukin 12-induced activation and interferon gamma secretion of peripheral natural killer T cells in nonobese diabetic mice suggests new pathogenic mechanisms for insulin-dependent diabetes mellitus.

The function of natural killer T (NKT) cells in the immune system has yet to be determined. There is some evidence that their defect is associated with autoimmunity, but it is still unclear how they play a role in regulating the pathogenesis of T cell-mediated autoimmune diseases. It was originally proposed that NKT cells could control autoimmunity by shifting the cytokine profile of autoimmune T cells toward a protective T helper 2 cell (Th2) type. However, it is now clear that the major function of NKT cells in the immune system is not related to their interleukin (IL)-4 secretion. In fact, NKT cells mainly secrete interferon (IFN)-gamma and, activated in the presence of IL-12, acquire a strong inflammatory phenotype and cytotoxic function.     

Resistance alleles at two non-major histocompatibility complex-linked insulin-dependent diabetes loci on chromosome 3, Idd3 and Idd10, protect nonobese diabetic mice from diabetes

Development of diabetes in NOD mice is polygenic and dependent on both major histocompatibility complex (MHC)-linked and non-MHC-linked insulin-dependent diabetes (Idd) genes. In (F1 x NOD) backcross analyses using the B10.H-2g7 or B6.PL-Thy1a strains as the outcross partner, we previously identified several non-MHC Idd loci, including two located on chromosome 3 (Idd3 and Idd10). In the current study, we report that protection from diabetes is observed in NOD congenic strains having B6.PL-Thy1a- or B10-derived alleles at Idd3 or Idd10. It is important to note that only partial protection is provided by two doses of the resistance allele at either Idd3 or Idd10. However, nearly complete protection from diabetes is achieved when resistance alleles are expressed at both loci. Development of these congenic strains has allowed Idd3 to be localized between Glut2 and D3Mit6, close to the Il2 locus.     

CD1-restricted NK T cells protect nonobese diabetic mice from developing diabetes

NK T cells are a unique subset of T cells that recognize lipid antigens presented by CD1d. After activation, NK T cells promptly produce large amounts of cytokines, which may modulate the upcoming immune responses. Previous studies have documented an association between decreased numbers of NK T cells and the progression of some autoimmune diseases, suggesting that NK T cells may control the development of autoimmune diseases. To investigate the role of NK T cells in autoimmune diabetes, we crossed CD1 knockout (CD1KO) mutation onto the nonobese diabetic (NOD) genetic background. We found that male CD1KO NOD mice exhibited significantly higher incidence and earlier onset of diabetes compared with the heterozygous controls. The diabetic frequencies in female mice showed a similar pattern; however, the differences were less profound between female CD1KO and control mice. Early treatment of NOD mice with alpha-galactosylceramide, a potent NK T cell activator, reduced the severity of autoimmune diabetes in a CD1-dependent manner. Our results not only suggest a protective role of CD1-restricted NK T cells in autoimmune diabetes but also reveal a causative link between the deficiency of NK T cells and the induction of insulin-dependent diabetes mellitus.     

A defect in tryptophan catabolism impairs tolerance in nonobese diabetic mice

The predisposition of nonobese diabetic (NOD) mice to develop autoimmunity reflects deficiencies in both peripheral and central tolerance. Several defects have been described in these mice, among which aberrant antigen-presenting cell function and peroxynitrite formation. Prediabetes and diabetes in NOD mice have been targeted with different outcomes by a variety of immunotherapies, including interferon (IFN)-gamma. This cytokine may be instrumental in specific forms of tolerance by virtue of its ability to activate immunosuppressive tryptophan catabolism. Here, we provide evidence that IFN-gamma fails to induce tolerizing properties in dendritic cells from highly susceptible female mice early in prediabetes. This effect is associated with impaired tryptophan catabolism, is related to transient blockade of the Stat1 pathway of intracellular signaling by IFN-gamma, and is caused by peroxynitrite production. However, the use of a peroxynitrite inhibitor can rescue tryptophan catabolism and tolerance in those mice. This is the first report of an experimental autoimmune disease in which defective tolerance is causally linked to impaired tryptophan catabolism.     

Male gonadal environment paradoxically promotes dacryoadenitis in nonobese diabetic mice.

Similar to pancreatic islets, submandibular glands are more rapidly infiltrated in female NOD mice than in males. The present comparative analysis of cellular infiltrations in lacrimal glands, however, revealed the opposite finding. At 12 wk of age, approximately 25% of male lacrimal tissue area is infiltrated, whereas age-matched female NOD mice still lack major signs of inflammation. T cells predominate in early stages of invasion, but B cells accumulate promptly in more advanced stages, and ultimately dominate over T cells. Dacryoadenitis is promoted by sex hormones, as suggested by the reduced infiltrations seen in orchidectomized NOD males (P < 0.01). It is also controlled by the local environment provided by the lacrimal tissue. Splenocytes from 4- and 20-wk-old female NOD mice cause massive lesions upon adoptive transfer into NOD male recipients while, conversely, female recipients develop barely any histological sign of infiltration, even after transfer of splenocytes from 20-wk-old donor males. These observations provide strong evidence for a dacryoadenitis-promoting role of male gonadal hormones in NOD mice, a finding that contrasts the known androgen-mediated protective effects on insulitis and submandibulitis in the same strain and on dacryoadenitis in other animal models of Sjögren's syndrome.     

白细胞介素10基因对非肥胖糖尿病鼠1型糖尿病发病率的影响

目的:探讨白细胞介素10(IL-10)基因治疗对非肥胖糖尿病(nonobesediabetic,NOD)鼠1型糖尿病发病率、胰岛炎的影响。方法:4周龄雌性NOD小鼠尾静脉快速注射IL-10表达质粒,注射Ringes液作为对照组。酶联免疫吸附实验(ELISA)检测血清中IL-10的表达,10周龄后每周测定血糖,25周龄HE染色观察未发病的NOD鼠胰岛炎程度。结果:处理组IL10的表达持续14d左右,糖尿病发病率为25%,明显低于对照组(71%)(χ2=5.57,P<0.05),且处理组中未发糖尿病的NOD鼠胰岛炎的严重程度亦低于对照组。结论:IL-10基因给药可以降低NOD鼠1型糖尿病发病率及胰岛炎严重程度。     

Effects of low-dose cyclosporine prophylaxis in nonobese diabetic mice

Among female nonobese diabetic mice, ketotic insulin-dependent diabetes mellitus (IDDM) develops spontaneously in 80% between 12 and 26 weeks of age. This condition resembles human type I diabetes. IDDM developed in 0 of 11 (0%) mice prophylactically treated with 10 mg of cyclosporine A (CyA) per kg s.c. every 4th day from 8 to 26 weeks of age; 8 of 10 (80%) of sex- and age-matched controls developed IDDM; 2 of 8 (25%) followed up to 5 months beyond the time of drug administration developed IDDM. The distribution of specific radioactivity ([3H]CyA) was used to calculate the concentrations of CyA in serum, blood cells and various organs. Serum values of CyA produced by radioimmunoassay were higher than those estimated by the [3H]CyA method. Pancreata of CyA-treated mice were histologically normal. Pancreata of control mice showed lymphocytic infiltration of the islets of Langerhans. Neither hepatotoxicity nor nephrotoxicity assessed by biochemical and histological data was detectable in CyA-treated mice. The insulin secretory capacity of trypan blue viable functional pancreatic islets isolated post-treatment; was significantly lower in controls than in CyA-treated mice; islet content of insulin was not statistically different between controls and CyA-treated mice. We conclude that low nontoxic doses of CyA abrogate completely the development of diabetes in the female nonobese diabetic mouse and abolish lymphocytic infiltration of the islets of Langerhans against which there is autoimmunity. The effect of CyA persists well past the duration of therapy.     

Abrogation of autoimmune diabetes in nonobese diabetic mice and protection against effector lymphocytes by transgenic paracrine TGF-beta1.

Paracrine effect of transforming growth factor-beta1 (TGF-beta1) on autoimmune insulitis and diabetes was studied by transgenic production of the active form of porcine TGF-beta1 (pTGF-beta1) in pancreatic islet (islet) alpha cells in nonobese diabetic (NOD) mice under the control of rat glucagon promoter (RGP) (NOD-RGP-TGF-beta1). None of 27 NOD-RGP-TGF- beta1 mice developed diabetes by 45 wk of age, in contrast to 40 and 71% in male and female nontransgenic mice, respectively. None of the NOD-RGP-TGF-beta1 mice developed diabetes after cyclophosphamide (CY) administration. Adoptive transfer of splenocytes of NOD-RGP-TGF-beta1 mice to neonatal NOD mice did not transfer diabetes after CY administration. Adoptive transfer of three types of diabetogenic lymphocytes to NOD-RGP-TGF-beta1 and nontransgenic mice after CY administration led to the lower incidence of diabetes in NOD-RGP-TGF-beta1 mice versus that in nontransgenic mice: 29 vs. 77% for diabetogenic splenocytes, 25 vs. 75% for islet beta cell-specific Th1 clone cells, and 0 vs. 50% for islet beta cell-specific CD8(+) clone cells, respectively. Based on these, it is concluded that autoimmune diabetes in NOD mice is not a systemic disease and it can be completely prevented by the paracrine TGF-beta1 in the islet compartment through protection against CD4(+) and CD8(+) effector lymphocytes.     

Dendritic cells transduced to express interleukin-4 prevent diabetes in nonobese diabetic mice with advanced insulitis

Our previous studies demonstrated that adoptive transfer of dendritic cells (DC) prevents diabetes in young nonobese diabetic (NOD) mice by inducing regulatory T(H)2 cells. In this report, as a means of treating NOD mice with more advanced insulitis, we infected DC with adenoviral vectors expressing interleukin (IL)-4 (Ad.IL-4), eGFP (Ad.eGFP), or empty vector (Ad psi 5). DC infected with any of the Ad vectors expressed higher levels of CD40, CD80, and CD86 molecules than uninfected DC and Ad.IL-4 DC produced IL-4 after lipopolysaccharide (LPS) and interferon (IFN)-gamma stimulation. Ad-infected DC efficiently stimulated allogeneic T cells, and cultures of T cells with Ad.IL-4 DC produced lower levels of IFN-gamma and marginally higher levels of IL-4. In vivo studies demonstrated that the Ad.eGFP DC trafficked to the pancreatic lymph nodes within 24 hr of intravenous administration, and could be visualized in the T cell areas of the spleen. The intrapancreatic IFN-gamma:IL-4 or IFN-gamma:IL-10 cytokine ratios were lower in 10-week-old mice treated with Ad.IL-4 DC, and these mice were significantly protected from disease. These results demonstrate, for the first time, that genetically modified DC can prevent diabetes in the context of advanced insulitis.     

Clinico-morphological characteristics of diabetic nephroangiopathy in insulin-dependent diabetes mellitus

The clinicofunctional characteristics of the kidneys were estimated, biopsy of the kidney and skin with an immunohistochemical study was performed and some immunity values considered in 17 patients with type I diabetes mellitus. The study was performed with respect to the degree of expression of the clinical signs of nephropathy including the preclinical stage, too. Three types of morphological changes in the kidney and skin were singled out. Initial tubuloglomerular disorders occurred at the preclinical stage of nephroangiopathy. The nature of changes in the skin coincided mostly with changes in the kidney lagging behind in some cases. The results confirmed the leading role of metabolic derangements in the genesis of microangiopathies and a necessity of a good compensation of diabetes for its prevention. The immunohistochemical and immunological findings did not make it possible to exclude the immune mechanisms in the development of diabetic nephroangiopathy.     

Interleukin 4 reverses T cell proliferative unresponsiveness and prevents the onset of diabetes in nonobese diabetic mice

Beginning at the time of insulitis (7 wk of age), CD4+ and CD8+ mature thymocytes from nonobese diabetic (NOD) mice exhibit a proliferative unresponsiveness in vitro after T cell receptor (TCR) crosslinking. This unresponsiveness does not result from either insulitis or thymic involution and is long lasting, i.e., persists until diabetes onset (24 wk of age). We previously proposed that it represents a form of thymic T cell anergy that predisposes to diabetes onset. This hypothesis was tested in the present study by further investigating the mechanism responsible for NOD thymic T cell proliferative unresponsiveness and determining whether reversal of this unresponsiveness protects NOD mice from diabetes. Interleukin 4 (IL-4) secretion by thymocytes from > 7-wk- old NOD mice was virtually undetectable after treatment with either anti-TCR alpha/beta, anti-CD3, or Concanavalin A (Con A) compared with those by thymocytes from age- and sex-matched control BALB/c mice stimulated under identical conditions. NOD thymocytes stimulated by anti-TCR alpha/beta or anti-CD3 secreted less IL-2 than did similarly activated BALB/c thymocytes. However, since equivalent levels of IL-3 were secreted by Con A-activated NOD and BALB/c thymocytes, the unresponsiveness of NOD thymic T cells does not appear to be dependent on reduced IL-2 secretion. The surface density and dissociation constant of the high affinity IL-2 receptor of Con A-activated thymocytes from both strains are also similar. The patterns of unresponsiveness and lymphokine secretion seen in anti-TCR/CD3- activated NOD thymic T cells were also observed in activated NOD peripheral spleen T cells. Exogenous recombinant (r)IL-2 only partially reverses NOD thymocyte proliferative unresponsiveness to anti-CD3, and this is mediated by the inability of IL-2 to stimulate a complete IL-4 secretion response. In contrast, exogenous IL-4 reverses the unresponsiveness of both NOD thymic and peripheral T cells completely, and this is associated with the complete restoration of an IL-2 secretion response. Furthermore, the in vivo administration of rIL-4 to prediabetic NOD mice protects them from diabetes. Thus, the ability of rIL-4 to reverse completely the NOD thymic and peripheral T cell proliferative defect in vitro and protect against diabetes in vivo provides further support for a causal relationship between this T cell proliferative unresponsiveness and susceptibility to diabetes in NOD mice.