Human Insulin-dependent Diabetes Mellitus Research Articles

Disease-associated Autoimmunity and Prevention of Insulin-dependent Diabetes Mellitus

Insulin-dependent diabetes mellitus (IDDM) is a chronic autoimmune disease with a subclinical prodromal period characterized by selective destruction of insulin-producing β cells in the pancreatic islets. This process is assumed to be T-cell mediated, but the emergence of disease-associated autoantibodies into the peripheral circulation is usually the first noticeable sign of β-cell autoimmunity in human IDDM. Recent observations have suggested that β-cell autoimmunity may be induced in any individual at any time. There are also data indicating that such autoimmunity may have been initiated prenatally in some individuals. Only a proportion of those with signs of islet cell autoimmunity progress to clinical disease, and harmless β-cell autoimmunity reflected by positivity for a single autoantibody specificity seems to appear without any relation to genetic IDDM susceptibility. One can hypothesize that in most subjects HLA-conferred protection against IDDM prevents the β-cell process from progressing to a stage of destructive insulitis that may lead to clinical disease. Environmental factors may trigger initial β-cell damage and subsequently accelerate the destructive process. Non-HLA genes may also be involved in the regulation of the progression rate. Prospective observations of prediabetic individuals have revealed that IDDM-associated autoantibodies tend to appear sequentially, and that those who progress to clinical disease mount a strong humoral immune response to most known disease-associated antigens. This indicates that the immune response associated with β-cell destruction is not purely T-heiper 1 biased. Antigen-specific immunotherapy may in the future offer effective measures to intervene in preclinical IDDM to prevent end-stage insulitis. Substantially more data have to be generated, however, on the mechanisms, efficacy and safety of such therapy before it is possible to judge its clinical applicability.

Genetics of type 1 insulin-dependent diabetes mellitus.

Many human insulin-dependent (type 1) diabetes mellitus (IDDM) genes have recently been mapped. The HLA region on chromosome 6 and the insulin gene promoter on chromosome 11p have been extensively investigated. Genome scanning of many families with several affected individuals has successfully mapped other predisposing loci situated on different chomosomal regions. In addition, the positional cloning of IDDM susceptibility genes is in progress. Advances in high throughput genotyping and large-scale sequencing, and the availability of high-density maps of human genes will help to identify IDDM genes. Understanding the genetic basis of IDDM will aid in the design of new strategies to prevent or cure the disease.

Beta-Cell Rest: A Strategy for the Prevention of Autoimmune Diabetes

An autoantigen being recognized by specific receptors is the key reaction of an autoimmune disease. Whereas much efforts have been made to develop immunosuppressive regimens which reduce the amount of effector cells, and/or inhibit receptor activation, surprisingly little attention has been paid to reduce the ligand-receptor interaction by interfering with the amount of antigen being presented from the target cells. In this review, we discuss clinical observations in autoimmune endocrine disease which illustrate that target cell alterations can modify the disease activity and comment on recent clinical trials which indicate that beta-cell rest may be beneficial to the course of human autoimmune diabetes mellitus.

Lack of Autoimmune Serological Reactions in Rodent Models of Insulin Dependent Diabetes Mellitus

Spontaneous insulitis with insulin-dependent diabetes mellitus (IDDM) in rodent models, the BB rat and NOD mouse, has clarified the pathogenesis of and guided decisions on interventional therapy for human IDDM. However, the occurrence in such models of a standard marker of human IDDM, autoantibodies to β islet cell constituents, has been controversial. Hence we assessed diabetes-prone rodents for the frequencies of raised levels of auto-antibodies to glutamic acid decarboxylase GAD (anti-GAD), insulin and heat shock protein 65 (HSP-65) in relation to levels in non-diabetes-prone animals and levels in human diabetic sera. Assays were performed sequentially at various ages of life. The immunoassays used for anti-GAD and anti-insulin were those validated for sensitivity and specificity for detection of the corresponding autoantibodies in human IDDM sera at international workshops. Positive controls included human IDDM sera with reactivity with GAD or insulin and, for mouse anti-GAD, the highly reactive monoclonal antibody, GAD-6. The results were that levels of autoantibodies in diabetes-prone BB rats or NOD mice to the ‘IDDM-relevant’ autoantigens in our panel did not exceed levels in control rats or mice, and were much lower than levels in humans with IDDM. We conclude that the BB rat and NOD mouse represent a model, but not a facsimile, of human IDDM and that therapeutic successes in such models should be interpreted with caution in relation to interventional therapy for human IDDM.

Effect of 5-alpha dihydrotestosterone on T-cell proliferation of the female nonobese diabetic mouse.

Nonobese diabetic (NOD) mice develop type I diabetes spontaneously and have been utilized as a model for human autoimmune insulin-dependent diabetes. The disease is caused by the destruction of insulin-producing beta cells in the pancreatic islet of Langerhans by infiltrating inflammatory cells, which are primarily T lymphocytes. The incidence of diabetes in NOD mice is increased in females compared with males, suggesting that sex steroid hormones play an important role in the development of the disease. We therefore investigated the effect of a male steroid, 5-alpha-dihydrotestosterone (5DHT), on disease development, T-cell phenotype, T-cell proliferation, and cytokine profiles in this model. None of the mice that received 5DHT for 120 days (n = 7) developed insulitis, whereas all control mice (n = 8) developed the disease. The percentage of CD4+ T cells in peripheral blood mononuclear cells was markedly decreased in the 5DHT-treated females compared with those in controls (37.1 +/- 4.8 vs 51.3 +/- 9.3, P < 0.02), whereas no significant differences in the percentage of CD8+ T cells were observed between treated and control female mice. Results of a syngeneic mixed lymphocyte reaction (SMLR) also suggested that T cells are major target cells of 5DHT administration. An increased expression of IL-4 mRNA, representing T helper 2 (Th2) T cells, was observed in the SMLR. On the basis of these results, a systemic administration of 5DHT appears to have direct effects on the expansion of Th2 cell populations with subsequent restoration of normal immune responses.

Aerosol insulin induces regulatory CD8 gamma delta T cells that prevent murine insulin-dependent diabetes.

Cellular immune hyporesponsiveness can be induced by the presentation of soluble protein antigens to mucosal surfaces. Most studies of mucosa-mediated tolerance have used the oral route of antigen delivery and few have examined autoantigens in natural models of autoimmune disease. Insulin is an autoantigen in humans and nonobese diabetic (NOD) mice with insulin-dependent diabetes mellitus (IDDM). When we administered insulin aerosol to NOD mice after the onset of subclinical disease, pancreatic islet pathology and diabetes incidence were both significantly reduced. Insulin-treated mice had increased circulating antibodies to insulin, absent splenocyte proliferation to the major epitope, insulin B chain amino acids 9-23, which was associated with increased IL-4 and particularly IL-10 secretion, and reduced proliferation to glutamic acid decarboxylase, another islet autoantigen. The ability of splenocytes from insulin-treated mice to suppress the adoptive transfer of diabetes to nondiabetic mice by T cells of diabetic mice was shown to be caused by small numbers of CD8 gamma delta T cells. These findings reveal a novel mechanism for suppressing cell-mediated autoimmune disease. Induction of regulatory CD8 gamma delta T cells by aerosol insulin is a therapeutic strategy with implications for the prevention of human IDDM.

Major histocompatibility complex class I molecule expression is normal on peripheral blood lymphocytes from patients with insulin-dependent diabetes mellitus.

Recent work from one laboratory has shown, in both nonobese diabetic mice and humans, an association between insulin-dependent diabetes mellitus (IDDM) and quantitative difference in MHC class I molecule expression. This reported decrease in MHC class I molecule expression is very controversial in the nonobese diabetic mouse model of IDDM, but to our knowledge, it has not been evaluated by another group in human IDDM. To evaluate this question, we studied 30 patients with IDDM and 30 age- and sex-matched normal controls. MHC class I molecule expression was measured by flow cytometry with conformational-dependent MHC class I mAbs. The mean antigen density of MHC class I molecule expression in IDDM vs. normal control is 454+/-34 vs. 440+/-28 for lymphocytes and 1,440+/-117 vs. 1,494+/- 117 for monocytes, both P > 0.05. Three conformational-dependent MHC class I antibodies showed consistent results. To estimate the biological variation of MHC class I molecule expression in normal controls, we also studied 10 age- and sex-matched normal control pairs. Using X +/-SD of the percentage difference of mean antigen density in the normal control pairs as our definition of normal, we found that 70% (21/30) of IDDM patients had normal, 13% (4/30) of IDDM patients had decreased, and 17% (5/30) of IDDM patients had increased MHC class I molecule expression on lymphocytes. All IDDM patients showed normal MHC class I expression on monocytes. In conclusion, we find that there is no consistent decrease in MHC class I molecule expression on either lymphocytes or monocytes from patients with IDDM. The MHC class I molecule expression observed in IDDM patients is largely within the expected biological variation of MHC class I molecule expression that has been observed in normal controls.

A rapid method to study heat shock protein 70-2 gene polymorphism in insulin-dependent diabetes mellitus.

To examine the role of DNA loci within the human leukocyte antigen (HLA) region and insulin-dependent diabetes mellitus (IDDM), we studied fine mapping of HSP70-2 gene. Polymerase chain reaction (PCR)-based genotyping was then developed and applied to type HSP70-2 in 59 patients with IDDM and 83 unrelated controls recruited from the inhabitants of northern Taiwan. Southern blot analysis revealed a diallelic PstI polymorphism of the HSP 70-2 gene, i.e., 9.6- and 8.5-kb alleles. The polymorphic site was mapped in the intragenic PstI sequences (nucleotides 1051-1056) of the HSP70-2 gene. PCR-based restriction fragment length polymorphism studies revealed that the frequency of the 8.5-kb allele was increased in IDDM (56.8%, vs. 40.4% in controls; p < 0.009), with a relative risk of 1.93 (95% confidence interval = 1.20-3.11). The genotypic frequencies of 9.6/9.6, 9.6/8.5, and 8.5/8.5 were 17.0, 52.5, and 30.5% for IDDM were different from those of controls (36.1, 47.0, and 16.9%, respectively; the homozygous 9.6/ 9.6 genotype was significantly decreased in the IDDM group, p < 0.02). In conclusion, we provide a simple, rapid, and nonradioactive method for HSP70-2 genotyping. Our data confirmed that the 8.5-kb allele of HSP70-2 was associated with IDDM susceptibility in the Taiwanese population.

Differential expression of ICAM-1 and LFA-1 versus L-selectin and VCAM-1 in autoimmune insulitis of NOD mice and association with both Th1- and Th2-type infiltrates.

The infiltration of pancreatic islets by mononuclear cells is the hallmark of the development of insulin dependent diabetes mellitus (IDDM) in the NOD mouse, an animal model for human IDDM. The aim, of this study was to correlate adhesion molecule expression with the degree of islet infiltration and to compare Th1- and Th2-driven islet inflammation. Cryostat sections of NOD mouse pancreata before and after diabetes development were analysed by semiquantitative immunohistochemistry. NOD mouse islets did not show the expression of ICAM-1, LFA-1, L-selectin and VCAM-1 prior to infiltration by mononuclear cells. Furthermore, islets with early stage insulitis (grade 1, periinsular location of small infiltrates) still were devoid of adhesion molecule expression. ICAM-1 and LFA-1 were first demonstrable in islets with strong periinsular infiltrates (insulitis grade 2) while L-selectin and VCAM-1 were only seen in islets with mild or strong intraislet infiltration (grade 3-4). Adhesion molecules were demonstrable in areas of macrophage and T-lymphocyte infiltrates but not in adjacent endocrine islet tissue. Islets of all infiltration stages contained Th2 lymphocytes (positive for IL-4). Substantial numbers of Th1 cells (positive for IFN-gamma, TNF-alpha, IL-2 and/or IL-2 receptor) were observed only after acceleration of diabetes development by a single injection of cyclophosphamide (250 mg/kg i.p.). Interestingly, the adhesion molecule expression pattern in islets with "Th1' versus "Th2 insulitis' was not different. In conclusion, the expression of adhesion molecules in islets during the development of autoimmune diabetes does not precede mononuclear infiltration but probably occurs in response to the activation of initial small infiltrates. ICAM-1 and LFA-1 expression is seen prior to L-selectin and VCAM-1. However, adhesion molecule expression during Th1 versus Th2 cell infiltration is very similar, suggesting similar adhesion molecule requirements of the two Th subsets.

The BB rat as a model of human insulin-dependent diabetes mellitus.

Use of the BioBreeding (BB) rat to model human insulin-dependent diabetes mellitus (IDDM) is useful in that characteristics of diabetes in the BB rat closely parallel those observed in human IDDM. Diabetic animals can be biopsied, autopsied, and bred to study the genetic basis of IDDM. The genetic, immunological, and environmental components of the disease can all be investigated under controlled conditions. Two inbred lines of BB have been used in the majority of published studies using this model system; these rats are designated as diabetes prone (DP-BB/Wor) and diabetes resistant (DR-BB/Wor). This unit presents two protocols for the diagnosis and prevention of spontaneous IDDM in DP-BB/Wor rats. Two alternate protocols are given for the induction of autoimmune disease in DR-BB/Wor rats and for the adoptive transfer of autoimmunity into histocompatible athymic WAG nu/nu recipient rats. Support protocols are also given for diagnosis of insulitis, treatment of diabetic rats with insulin, and serological analysis of blood samples from sentinel rats to monitor the pathogen status of the colony.

Restoration of immune abnormalities in diabetic BB rats after pancreas transplantation. I. Macrochimerism of donor-graft-derived RT6+ T cells responsible for restoration of immune responsiveness and suppression of autoimmune reaction.

Diabetes-prone (DP) BB rats (RT1(u), RT6.1) spontaneously develop insulin-dependent diabetes mellitus (IDDM) and the disease manifestation resembles that in human IDDM. DP rats are immunodeficient with severe T lymphocytopenia due to the absence of T cells expressing the RT6 differential alloantigen, which have immunoregulatory functions. MHC- and non-MHC-compatible Wistar Furth (WF; RT1(u), RT6.2) pancreases were transplanted into DP rats. WF pancreas grafts were destroyed by IDDM recurrence (insulitis), but not by rejection, with a mean survival time of 65.3 +/- 21.7 days. To prevent the recurrence of IDDM in the grafts, monoclonal antibodies to intercellular adhesion molecule-1 and leukocyte function-associated antigen-1 were administered. WF pancreas grafts were indefinitely accepted (>108.0 +/- 26.8 days) in monoclonal antibody-treated DP recipients. The number of T cells was increased and cellular immune responses restored only in the DP rats that had accepted grafts. The increased number of T cells was due to the peripheral appearance of donor-type RT6.2+ T cells, which represented 34.3 +/- 7.0% of total splenic T cells. The cytotoxicity of splenic T cells to WF islet cells was suppressed in the presence of RT6+ T cells in vitro. These findings demonstrated that stable macrochimerism of donor-derived RT6+ T cells could restore the immune responses and prevent the recurrence of IDDM in the DP recipients.

Evidence that Th1 lymphocytes predominate in islet inflammation and thyroiditis in the BioBreeding (BB) rat.

Th1 cytokines are thought to play a key role in islet inflammation and destruction in insulin-dependent diabetes mellitus (IDDM). We studied this hypothesis in the diabetes-prone (DP)-BB and the diabetes-resistant (DR)-BB rats that are used as a model of human IDDM. The DP-BB rat develops spontaneous autoimmune diabetes at the age of 11-14 weeks. In the DR-BB rat, diabetes is inducible by depletion of RT6+ lymphocytes and coadministration of polyinosinic:polycytidylic acid (Poly I:C). We used reverse transcriptase-polymerase chain reaction (RT-PCR) and semi-quantitative PCR techniques to examine mRNA expression of Th1 and Th2 cytokines in inflamed islets and thyroids from DP-BB and DR-BB rats. We observed that in DP-BB and in treated DR-BB rats, the levels of TCR beta, IFN-gamma and IL-12p40 mRNA increase with disease progression. In contrast, expression of message for IL-2 and IL-4 is minimal to undetectable in DP-BB and RT6-depleted DR-BB animals at any age. Message for IL-10 is detectable in DP and DR islets; however, its level of expression does not change with disease progression. A similar cytokine mRNA profile is observed in inflamed thyroids from acutely diabetic RT6-depleted DR-BB rats. Incubation of 10 wk old DP islets for 48 h in the presence of anti-CD3 antibody, followed by an incubation with rIL-2 for an additional 5-7 days, results in an expansion of T lymphocytes, and these cells express high levels of IFN-gamma and IL-10 mRNA. Our results suggest that autoimmunity in DP-BB and DR-BB rats is mediated by Th1 lymphocytes and that IFN-gamma and IL-12 are likely to play a key role in islet and thyroid inflammation and destruction in IDDM.

Islet amyloid polypeptide (IAPP) secretion from pancreatic islets isolated from non-obese diabetic (NOD) mice

The secretion of islet amyloid polypeptide (IAPP) during the course of insulin-dependent diabetes mellitus (IDDM) is essentially unknown. In the present study we elucidated this issue by examining IAPP and insulin secretion from isolated pancreatic islets obtained from IDDM-prone female NOD mice aged 6–9 weeks and 12–15 weeks and from non-IDDM-prone male NMRI mice. Basal islet hormone secretion at 1.7 mM glucose and after stimulation with 17 mM glucose or with 17 mM glucose + 5 mM theophylline was studied acutely or after 1 week of tissue culture. The levels of glucose-stimulated insulin release from NOD mouse islets increased after tissue culture, whereas it remained unchanged in NMRI mouse islets. Overall changes in islet insulin secretion were accompanied by similar changes in IAPP secretion. Acute after isolation, islets from NMRI mice displayed a reduced IAPP/insulin secretion ratio in response to the stimulation, which could reflect a destabilized hormone release. When the NOD mouse islets at 6–9 weeks of age were exposed to secretory stimuli the molar ratio of IAPP/insulin secretion declined. At a later stage of advanced insulitis (12–15 weeks) also the basal IAPP/insulin secretory ratio at low glucose tended to decline. If extrapolated to the early prediabetic phase of human IDDM, this would mean that a relative hypersecretion of insulin in relation to IAPP might occur, due to an increased secretory demand for insulin or due to an intrinsic change in the biology of the secretory cells.

Immune regulation in type 1 diabetes.

The non-obese diabetic (NOD) mouse is an animal model of insulin-dependent diabetes mellitus (IDDM) that shows many of the characteristics of human IDDM. In the NOD model, there exists a discrepancy between the onset of insulitis and diabetes suggesting the potential existence of some form of immune regulation that delays beta cell destruction. Our transfer system using NOD-scid/scid (NOD-scid) mice as recipients of donor NOD cells suggested that immune regulatory cells exist in the periphery of NOD mice, not in the islets. These regulatory cells are considered to be memory CD4+ cells which show a Th2 (or Th zero) type cytokine profile following activation in vitro. The function of the memory CD4+ cells seems to change from protective to pathogenic as the disease progresses. Moreover, cytokine profiles of this CD4+ CD45RBlow (memory) population shifted from a Th2 (or Th zero) to a Th1 type response coincident with the onset of hyperglycaemia. These data suggest that the progression of NOD disease from insulitis to frank hyperglycaemia is under the control of CD4+ CD45RBlow immune 'regulatory' cells.

HLA and glutamic acid decarboxylase in human insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is linked to HLA factors on human chromosome 6 and strongly associated with the presence of autoantibodies against the glutamic acid decarboxylase isoform GAD65. These autoantibodies, GAD65Ab are detected both before and at the time of clinical diagnosis. Molecular sequencing of HLA alleles and PCR-based genotyping have improved our understanding of the linkage between HLA and IDDM. At the same time, the molecular cloning of human islet GAD65 and the development of precise and reproducible GAD65Ab assays with recombinant human GAD65 has given new insights to the problem of to what extent HLA control the development of a GAD65 immune response or to the development of IDDM. Recent data are briefly reviewed. In new onset IDDM patients GAD65Ab were associated with the DQ2/8 or DQ2/X genotype. However, in patients with an older age at onset the association was particularly pronounced with the DQ2/8 genotype. The DQ5/8 genotype was significantly decreased among GAD65Ab positive patients. Certain DQ genotypes, therefore, seem permissive for the formation of GAD65Ab in IDDM. Studies of the general population is needed to determine if the DQ2, 8 or both alleles predispose to GAD65 autoreactivity. This is important since other factors may control the development of IDDM in only a fraction of GAD65 antibody positive individuals detected following a screening of the general population.

Vitamin A status affects the development of diabetes and insulitis in BB rats

BB/Wor rats develop autoimmune diabetes mellitus with many features in common with human insulin-dependent diabetes mellitus. Since retinoids are known to have effects on insulin secretion and immune function, these studies were designed to investigate the effects of retinoid deficiency on diabetes in BB/Wor rats and to identify a role for retinoid status in the pathogenesis of autoimmune diabetes mellitus. Litters of diabetes-prone (DP) and diabetes-resistant (DR) BB/Wor rats were divided at weaning and fed a diet either (1) devoid of retinoids and leading to clinical deficiency at approximately 60 days of age (A-def diet)—following 10 days of clinical deficiency, rats on the A-def diet were changed to a diet containing 2 μg/g retinoic acid (A-def/RA diet); (2) containing 2 μg/g retinoic acid but deficient in retinol (RA diet); or (3) replete in retinol with 4 μg/g retinyl palmitate (RP diet). Rats receiving RP or RA diets were pair-fed to rats on the A-def/RA diet. Diabetes by 120 days of age was greatly reduced ( P < .01) in DP rats that received the A-def/RA diet (four of 27) or RA diet (four of 29) versus the RP diet (13 of 31). Insulitis progressed with age in nondiabetic DP rats receiving the RP diet ( P < .02) or RA diet ( P < .05), but not the A-def/RA diet ( P > .22). Insulin secretion was measured in perfused pancreas of nondiabetic rats after age 120 days and correlated negatively with insulitis ( P < .05). DP rats receiving the RP diet had reduced insulin secretion as compared with other DP and DR rats ( P < .05). In DR rats, retinoid status had no effects on insulitis through 120 days of age or on insulin secretion after 120 days of age. In conclusion, retinol deficiency reduces diabetes and insulitis in DP BB/Wor rats, and retinoic acid can at least partly substitute for retinol in the development of insulitis.

T-cell recognition of β-cell autoantigens in insulin-dependent diabetes mellitus

Autoimmune T cells reactive to beta-cell autoantigens are generally believed to play an essential role in the immune-mediated selective pancreatic islet beta-cell destruction process leading to insulin-dependent diabetes mellitus (IDDM). Many of the supportive data have been obtained from animal models of this disease, but often these data remain to be validated in human IDDM, including the nature of the responsible autoreactive T cells and their targets on the beta cells. In the last few years, however, considerable progress has been made, and several candidate autoantigens have been identified. Diabetogenic T-cell clones have been isolated and characterized in animal models, but for the majority of these clones, the target autoantigen is unknown. In humans, the first islet autoantigens recognized by autoreactive T cells have been defined. This opens the way to designing immunointerventive strategies selective for these T cells and their candidate target antigens, in an attempt to prevent the onset of IDDM. In this review, we described the significance of T lymphocytes for the pathogenic process leading to type 1 diabetes and our studies showing (auto)immune responses by beta-cell-reactive T lymphocytes of newly diagnosed patients.

Quantitative immunohistochemical changes in the endocrine pancreas of nonobese diabetic (NOD) mice.

The nonobese diabetic (NOD) mouse is an animal model that shares a number of clinical, genetic, and immunologic characteristics with human insulin-dependent diabetes mellitus. Since little is known about the morphometric cell profiles in the endocrine pancreas of these NOD animals, it was of interest to assess their changes in morphometry within the pancreatic islet cell types during two stages of this syndrome. Prediabetic (6-week-old) and diabetic (16-week-old) NOD female mice, as well as normal C57BL/6 female mice (15 weeks old), were used. Light microscopic immunocytochemical and morphometric methods were employed to study the endocrine cell populations. The immunoperoxidase technique for the identification of insulin, glucagon, somatostatin, and pancreatic polypeptide, as well as the point-counting method, was used on serial sections of pancreas tissue. Compared to those of normal and prediabetic mice, pancreata from diabetic animals showed a decrease in both the number of islets and the volume density of the endocrine component. Analysis of islet tissue revealed a significant diminution of B-cell volume density, as well as an increased A-, D-, and PP-cell volume density. A parallel variation in the number of B and non-B cells was also found. In addition, when the total pancreatic tissue surface was taken as reference, the fractional area occupied by all the different types of islet cells was seen to be diminished in a variable fashion. We conclude that the diabetic syndrome of NOD mice not only severely affects the B-cell mass, but also causes marked changes in the non-B endocrine-cell populations.

A major histocompatibility complex class II restriction for BioBreeding/Worcester diabetes-inducing T cells.

Inbred diabetes-prone (DP) BioBreeding/Worcester (BB/Wor) (RT1u) rats develop spontaneous autoimmune diabetes, which, like human insulin-dependent diabetes mellitus, is mediated by autoreactive T lymphocytes. Breeding studies have shown an absolute requirement for at least one copy of the major histocompatibility complex (MHC) RT1u haplotype for spontaneous diabetes expression. Concanavalin A-activated spleen cells from acutely diabetic DP rats adoptively transfer diabetes only to recipients that express at least one RT1u haplotype. To investigate the basis for the MHC requirement in BB/Wor autoimmunity, diabetes-inducing T cell lines were derived from the spleens of acutely diabetic DP rats. Upon activation in vitro with islet cells, the T cell lines adoptively transfer insulitis and diabetes into young DP recipients and non-diabetes-prone RT1 congenic rat strains that are class IIu. Recipients that are RT1u at only the class I A or C locus, but not at the class II B/D loci, do not develop diabetes after T cell transfer. The adoptive transfer of diabetes by Concanavalin A-activated diabetic DP spleen cells also requires that donor and recipient share class II B/Du gene products. Furthermore, the adoptive transfer of diabetes into MHC class IIu congenic rats is independent of the class I haplotype; i.e., it occurs in the presence of class I Aa Cu or Au Ca gene products. BB/Wor T cells can be activated in vitro for the transfer of diabetes with islet cell antigens and class II-positive but not class IIu-negative antigen-presenting cells. The inductive phase of BB diabetes is therefore MHC class II restricted, and this appears to operate at the level of interaction between inducing T cells and class IIu antigen-presenting cells. These results may explain the well-documented, but not yet understood, MHC class II genetic contribution to insulin-dependent diabetes mellitus pathogenesis, and they may facilitate the development of protocols designed to prevent diabetes onset in susceptible individuals.

Low bcl-2 expression and increased spontaneous apoptosis in T-lymphocytes from newly-diagnosed IDDM patients

The bcl-2 gene product has been shown to regulate apoptotic cell death, and its dysregulation has been shown to induce several abnormalities in the immune system. No data exist regarding bcl-2 expression in autoimmune diseases, such as human insulin-dependent diabetes mellitus (IDDM). We investigated bcl-2 protein expression by testing T lymphocytes from 15 newly-diagnosed (<3 weeks) IDDM patients in comparison to 10 age-matched control subjects. The expression of bcl-2 on CD3+ lymphocyte subsets was investigated after membrane permeabilization by two- or three-colour immunofluorescence. When the percentage and mean fluorescence intensity (MFI) of bcl-2+/CD3+ cells from normal individuals and patients were compared, we found that bcl-2 expression within the CD3+ and CD4+ CD45R0+ T-cell populations was reduced significantly in IDDM patients (46.8±15.4 vs 79.6±11.7; 25.7±3.8 vs 47.15±5.7, respectively; p<0.001). To establish whether low bcl-2 expression in T cells from newly-diagnosed patients reflects their susceptibility to death by an apoptotic process, we also evaluated DNA staining with propidium iodide in CD3+ lymphocyte suspension after a (24–72 h) culture period (spontaneous apoptosis). We found that IDDM patients have higher levels of spontaneous apoptosis (mean±SEM: 24 h=4.6±0.8; 48 h=9.9±1; 72 h=12.8±1.1) than control subjects (24 h=1.8±0.4; 48 h=4.6±0.4; 72 h=5.7±0.3; p<0.02–0.001). Our study suggests that recent onset IDDM is characterised by reduced bcl-2 expression, which in turn may be associated with the increased spontaneous apoptosis we observed.