Development of autoimmune insulitis is prevented in E alpha d but not in A beta k NOD transgenic mice

Two lines of E alpha d-expressing NOD mice were established by continuously backcrossing [E alpha d B6 transgenic mice x NOD] F1 to parental NOD or directly microinjecting the E alpha d gene into fertilized NOD eggs. Similarly, A beta k-expressing transgenic NOD mice were produced. Subsequent histological examination of pancreatic tissues revealed that autoimmune insulitis was prevented in E alpha d backcross and transgenic mice but not in A beta k transgenic mice.     

Overexpression of bcl-2 in T cells affects insulitis in the nonobese diabetic mouse

The nonobese diabetic (NOD) mouse is a useful model for human autoimmune diabetes. The gene for the anti-apoptotic protein Bcl-2 has previously been suggested as a probable susceptibility candidate for the NOD mouse disease. In this study, we investigated how overexpression of Bcl-2 in lymphocytes might affect insulitis in NOD mice. A bcl-2 transgene expressed constitutively under the SV40-promoter and the 5'Igh enhancer, Emu, was bred onto NOD background. Two bcl-2 transgenic NOD strains were produced and analysed, one with overexpression of Bcl-2 on only B cells and the other with overexpression of Bcl-2 on both B and T cells. Subsequent to verification of expression pattern and functionality of the transgene, insulitis intensity was investigated in different backcross generations of the two transgenic strains. Overexpression of Bcl-2 on both B and T cells leads to a statistically significant protection of the mice from insulitis compared with normal littermates. Overexpression of Bcl-2 on only B cells, on the other hand, does not have any statistically significant effect on insulitis. Possible mechanisms for the effect of Bcl-2 on insulitis in NOD mice are discussed.     

Quantitative analysis of integrated Ed alpha gene expression in C57BL/6 transgenic mice.

We performed quantitative analysis of Ed alpha gene expression in the transgenic mice, created by microinjecting cloned Ed alpha gene fragments into C57BL/6 fertilized eggs. DNA dot-blot analysis revealed that Ed alpha gene-introduced transgenic mice (B6Ed alpha transgenic mice) contain 20 copies per cell of the Ed alpha gene in their genome. RNA dot-blot analysis revealed that the amount of Ed alpha mRNAs in B6Ed alpha transgenic spleen cells is 20-40-fold higher than those in normal BALB/c or (BALB x C57BL/6)F1 (CBF1) spleen cells. However, the amount of Ed alpha molecules expressed on B6Ed alpha transgenic spleen cells was similar to that expressed on normal BALB/c of CBF1 spleen cells on a gene-dose basis. The amount of endogenous Ed alpha mRNA in the B6Ed alpha transgenic spleen cells was almost equal to that of normal B6 spleen cells. Since the cell surface I-E molecule is formed by non-covalent association of E alpha and E beta chain, these results suggest that, in spite of the high expression of integrated Ed alpha gene in the cytoplasm of B6Ed alpha transgenic mice, the amount of Ed alpha gene expression on the cell surface is limited by the amount of endogenous Eb beta gene products.     

Neonatal Pattern of VH Gene Utilization in Nonobese Diabetic Mice Does not Correlate with Development of Insulitis

The nonobese diabetic (NOD) mouse model is a model of human autoimmune insulin dependent diabetes, IDDM. The effector cells of the disease have been shown to be T cells, but also B cells seem to contribute. Adult NOD mice have been shown to display a bias in their utilization of immunoglobulin (Ig) variable heavy (V(H)) genes. In this study the analysis of VH gene utilization in NOD mice protected from insulitis by transgenic insertion of a major histocompatibility complex (MHC) class II E(alpha) gene, point out that the bias in V(H) gene expression is not correlated to disease development. The aberrant V(H) gene utilization pattern in mice with the NOD genetic background is instead suggested to be a consequence of a deregulation of the apoptosis inhibiting gene bcl-2. We also investigated if prolonged in vitro survival of NOD lymphocytes is correlated to disease development. The E(alpha) transgenic NOD mice were shown to display a prolonged in vitro survival of spleen T cells, similar to normal NOD mice. These results indicate that defective death mechanisms of T cells may not be primarily involved in the development of autoimmune disease in these mice. However, in contrast to results from other groups, no difference in in vitro survival could be detected for B cells from mice with NOD genetic background compared to C57BL/6 mice.     

Adoptive transfer of islet antigen-autoreactive T cell clones to transgenic NOD.Ea(d)mice induces diabetes indicating a lack of I-E mediated protection against activated effector T cells

Transgenic insertion of the MHC class II Ea(d)gene in NOD mice restores I-E expression and prevents T-cell-mediated autoimmune diabetes (IDDM). The specific molecular and cellular mechanisms responsible for the diabetes resistance of transgenic NOD.Ea(d)mice remain unclear. We adoptively transferred islet antigen-specific T cell clones into NOD and transgenic NOD.Ea(d)mice to evaluate the level of protection provided by I-E expression against activated effector T cells. We have found that neither neonatal or 3-5-week-old I-E-expressing NOD.Ea(d)mice can completely inhibit the diabetogenic activities of activated islet antigen-specific T cell clones. These data indicate that Ealpha protein expression in NOD antigen presenting cells (APC) does not reduce islet autoantigen presentation in the context of I-A(g7)below the threshold required for stimulation of effector/memory diabetogenic T cells. Our results suggest that the mechanism of Ealpha protein-mediated diabetes resistance in NOD mice may be "antigen ignorance," in which the quantity of islet autoantigens presented in the context of I-A(g7)by APC is reduced below the threshold required to activate nai;ve islet antigen-specific T cells.     

Effects of BCG, lymphotoxin and bee venom on insulitis and development of IDDM in non-obese diabetic mice

To investigate whether BCG, lymphtoxin (LT) or bee venom (BV) can prevent insulitis and development of diabetes in non-obese diabetic (NOD) mice, we measured the degree of insulitis and incidence of diabetes in 24 ICR and 96 female NOD mice. NOD mice were randomly assigned to control, BCG-, LT-, and BV-treated groups. The BCG was given once at 6 weeks of age, and LT was given in 3 weekly doses from the age of 4 to 10 weeks. The BV was injected in 2 weekly doses from the age of 4 to 10 weeks. Diabetes started in control group at 18 weeks of age, in BCG group at 24 weeks of age, and in LT- or BV-treated group at 23 weeks of age. Cumulative incidences of diabetes at 25 weeks of age in control, BCG-, LT-, and BV-treated NOD mice are 58, 17, 25, and 21%, respectively. Incidence and severity of insulitis were reduced by BCG, LT and BV treatment. In conclusion, these results suggest that BCG, LT or BV treatment in NOD mice at early age inhibit insulitis, onset and cumulative incidence of diabetes.     

Local production of human IL-6 promotes insulitis but retards the onset of insulin-dependent diabetes mellitus in non-obese diabetic mice

We produced transgenic mice which overexpress human IL-6 inpancreatic ß cells of C57BL/6xCBA and non-obese diabetic(NOD) mice. Transgenic C57BL/6xCBA mice back-crossed onto aC57BL/6 background do not develop insulitis or diabetes. Incontrast, NOD/F₁ transgenic mice develop a lymphocytic infiltrateof pancreatic islets which is not seen in negative littermates.Immunohlstochemistry reveals these cells to be predominantlyCD4⁺, CD8⁺, B220⁺ cells. Despite the insulitis, these mice donot develop diabetes. Transgenic rat insulin promoter-IL-6 micewere therefore also made on an inbred NOD background. Thesemice showed no difference in the onset or extent of insulitiswhen compared with non-transgenic NOD mice and no differencewas found in the phenotype of the infiltrating cells. However,transgenic NOD mice had lower average fasting glucose levelsand delayed onset of diabetes compared with age and sex matchedlittermate negative NOD mice. As a consequence, transgenic NODmice also had longer survival than littermate negative NOD mice.We conclude that the expression of IL-6 byß cellsdoes not cause insulitis or diabetes in C57BL/6xCBA mice, butthat the interaction of IL-6 and diabetes susceptibility genescauses insulitis in NOD/F₁ mice. Since IL-6 delays the onsetof diabetes and prolongs survival of NOD mice, it is possiblethat the protective effect is caused by local IL-6 action onthe islets, by the infiltrating lymphocytes or both.     

No Evidence for TCR Vβ Repertoire Changes Influencing Disease Protection in E-Transgenic NOD Mice

In order to study whether positive selection of T cells plays any role in the MHC-dependent protection from diabetes in the non-obese-diabetic (NOD) mouse, the T cell Vβ repertoire has been studied in NOD mice and in NOD mice either transgenic for the wildtype MHC class II Eα gene, or for ΔY, a promotor-mutagenized Eα gene with a restricted tissue expression. The Eα transgenic line is protected from both insulitis and diabetes. The ΔY transgenic line is neither protected from insulitis nor from diabetes, although it can perform both positive and negative E-mediated selection in the thymus. The Vβ repertoire was studied in the pancreatic lymph nodes as these drain the area which is the target for the autoimmune attack. We see no evidence for Ea TCR Vβ repertoire differing from both nontransgenic NOD mice and ΔY mice despite its striking difference in susceptibility to autoimmunity. We conclude that none of the differences in the TCR Vβ repertoire of Eα-transgenic NOD mice hitherto observed are likely to explain the protective effect of E molecule expression in NOD mice.     

Non-tolerance and differential susceptibility to diabetes in transgenic mice expressing major histocompatibility class II genes on pancreatic beta cells

In insulin-dependent diabetes mellitus aberrant expression of major histocompatibility complex (MHC) class II antigens might induce autoimmune destruction of insulin-producing pancreatic beta cells. Here we demonstrate that already the expression of single MHC class II chains (E alpha k and E beta b) is sufficient to cause diabetes without affecting beta cell morphology and cell numbers. Our transgenes interfere at least in two points of insulin production leading to a severely diabetic phenotype. In one case E beta b expression led to a 10-30-fold decrease of mouse insulin mRNA. In another case E alpha k expression reduced insulin secretion to background levels. In addition, we also found a patchy distribution of both insulin and E alpha k expression, indicating heterogeneity of the beta cell population, without the concomitant development of diabetes. Although the transgenic E alpha k E beta b MHC class II molecules were expressed on the surface of pancreatic beta cells, the transgenic mice did not prove to be tolerant for I-E antigen. Autoimmune reactions remained absent showing that aberrant MHC class II expression on pancreatic beta cells alone is not sufficient for the development of autoimmune diabetes in mice.     

The effect of bone marrow and thymus chimerism between non-obese diabetic (NOD) and NOD-E transgenic mice,on the expression and prevention of diabetes

The non-obese diabetic (NOD) mouse is an established animal model of the autoimmune disease, insulin-dependent diabetes mellitus (IDDM). The NOD-E mouse is a transgenic mouse which expresses the I-E molecule (absent in NOD mice). Expression of I-E protects these mice from both insulitis and IDDM.We have investigated the possible mechanisms of this protection by constructing bone marrow, and combined bone marrow and thymus chimeras between NOD and NOD-E mice. Our data suggest that thymic epithelium may play no direct role in either protection against, or promotion of, IDDM. Protection from diabetes is provided either by NOD-E donor bone marrow or NOD-E recipient non-thymic radioresistant cells. The means by which protection may be achieved in this system are discussed.     

Short administration of polyclonal anti-T cell antibody (ALS) in NOD mice with extensive insulitis prevents subsequent development of autoimmune diabetes

Treatment of overtly diabetic NOD mice with antilymphocyte serum (ALS), a polyclonal anti-T cell antibody, leads to cure of diabetes. Here, we investigated whether ALS-treatment of NOD mice after development of extensive insulitis prevents onset of diabetes. Female NOD mice were treated with two doses of ALS at 14, 19 or 23 weeks of age. No further treatment was given. In untreated female NOD mice, diabetes developed starting at 13 weeks and reached 68% by 37 weeks. ALS-treatment at 14, 19 or 23 weeks when histology showed progressive insulitis completely prevented onset of overt diabetes in 9/12, 11/12 or 12/12 mice, respectively. Intraperitoneal glucose tolerance tests in 43 week-old ALS-treated, diabetes-free mice showed a normal pattern. Co-adoptive transfer of lymphoid cells prepared from ALS-treated diabetes-free mice together with splenocytes from overtly diabetic NOD mice resulted in marked delay in diabetes onset in NOD.SCID mice, suggesting the presence of autoimmune regulatory cells in ALS-treated mice. Autoimmune regulatory cells were CD4(+)CD25(+), but not CD4(+)CD25(-), T cells. Thus, treatment of euglycemic individuals who already show signs of autoimmune diabetes with a short course of polyclonal anti-T cell antibody may effectively prevent onset of type 1 diabetes mellitus.     

Direct evidence for the contribution of B cells to the progression of insulitis and the development of diabetes in non-obese diabetic mice

The non-obese diabetic (NOD) mouse is an excellent animal model of autoimmune diabetes associated with insulitis. The progression of insulitis causes the destruction of pancreatic beta cells, resulting in the development of hyperglycemia. Although it has been well documented that T cells are required for the development of insulitis and diabetes in NOD mice, the importance of B cells remains unclear. To clarify the role of B cells in the pathogenesis of NOD mice, we therefore generated B cell-deficient NOD (B-NOD) mice. Surprisingly, none (of 13) of the B- NOD mice developed diabetes by 40 weeks of age, while the control littermates with B cells (B+NOD) suffered from a high proportion (43 of 49) of diabetes. The insulin reactivity of B+NOD mice was significantly impaired, while the B-NOD mice showed a good insulin response, thus suggesting the pancreatic beta cell function to be well preserved in B- NOD mice. Although B-NOD mice did develop insulitis, the extent of insulitis was significantly suppressed. These data thus provide the direct evidence that B cells are essential for the progression of insulitis and the development of diabetes in NOD mice.      

Inhibition of diabetes in non-obese diabetic mice by nicotinamide treatment for 5 weeks at the early age.

To know the effects of nicotinamide (NCT) treatment for 5 weeks at the early age on insulitis and development of diabetes in non-obese diabetic (NOD) mice, this experiment was performed. Ten ICR (Institute of Cancer Research) and 15 female NOD mice at 4 weeks of age were used. Mice were assigned to ICR and NOD groups, and NOD mice were randomly divided to control and NCT-treated groups. NCT was administered to mice orally as a solution and in a dose of 500 mg/kg body weight a day from the age of 4 to 8 weeks. Diabetes onset was 18 weeks of age in control group, and 22 weeks of age in NCT-treated group. Cumulative incidences of diabetes at 25 weeks of age in control and NCT-treated NOD mice were 63 and 29%, respectively. Insulitis occurred in all NOD mice. Incidence of insulitis in total islets was decreased by NCT treatment in diabetic NOD mice, but intensity of insulitis was not improved by NCT treatment. Blood glucose level was increased markedly, and plasma insulin level was decreased by diabetes development in NOD mice. Plasma triglycerides and total cholesterol levels were increased in diabetic mice than in non-diabetic mice. In conclusion, these results suggest that NCT treatment for 5 weeks at the early age in NOD mice inhibits development of diabetes and insulitis in diabetic NOD mice.     

Role of MHC, Mls and TCR in immune tolerance

MHC class II molecules and self antigens, such as Mls, influence T-cell selection by clonal deletion of potentially self-reactive T cells. In order to examine the role of various class II molecules in the T-cell receptor-self antigen interaction, class II transgenic and recombinant mice were analysed for TCR expression. Our studies indicate that the A alpha and E alpha chains can present Mls gene products for the clonal deletion of V beta 6-bearing T cells, and that the A alpha q chain is defective in this process. We have also shown that E alpha A beta heterodimer in transgenic and recombinant mice is expressed and functions to delete I-E reactive V beta llT cells, demonstrating again the role of the E alpha molecule.     

Disease-protected major histocompatibility complex Ea-transgenic non-obese diabetic (NOD) mice show interleukin-4 production not seen in susceptible Ea-transgenic and non-transgenic NOD mice.

The non-obese diabetic (NOD) mouse is an animal model for insulin-dependent diabetes that has many similarities to the human disease. NOD mice transgenic for the Ea gene, allowing expression of the E molecule, are protected from diabetes and rarely develop insulitis. An Ea transgene mutated in the promoter region, (DeltaY) lacks E expression on most B cells, thymic medullary epithelium and primary antigen-presenting cells, and confers no protection whatsoever. We have used these transgenic NOD mice, together with non-transgenic NOD mice, to study the correlation of E expression and production of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma). We show that protected E-transgenic NOD mice have elevated levels of IL-4 compared with non-transgenic mice, both in the thymus and in the periphery. However, susceptible DeltaY-transgenic mice have elevated thymic IL-4 levels, but express almost as little IL-4 as non-transgenic NOD mice in the periphery. This drop in peripheral IL-4 production seen in DeltaY-transgenic mice thus correlates with the decreased E expression in the periphery of DeltaY-transgenic NOD mice. In contrast, there were no differences in IFN-gamma production between the three NOD lines. We suggest that Ea-transgenic NOD mice have E-selected regulatory T cells producing IL-4, which are subsequently activated by E-expressing primary antigen-presenting cells in the periphery. This activation would then be instrumental for the E-mediated protection from disease in NOD mice. Such a process would explain the total absence of protection in DeltaY-transgenic NOD mice, despite their widespread E expression.     

Immunobiology of DC in NOD mice.

NOD mice spontaneously develop diabetes between 15 and 20 weeks of age, which is preceded by insulitis characterized by the infiltration of lymphocytes. Dendritic cells (DC) are among the first cells to infiltrate the islet and they have been implicated in the pathogenesis of the disease. Our work has been concerned with the detailed characterization of four distinct DC populations in NOD mice: two derived from bone marrow (BM) cells cultured in either granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4) or GM-CSF alone and two from the spleen of Flt3 ligand (Flt3L) -treated mice, isolated on the basis of CD8alpha expression. Phenotypic and functional differences between these DC subsets in NOD mice have been identified. In addition, we obtained a lower yield of NOD BM-derived DC and they expressed higher levels of cell-surface CD40 and IL-12 p40 mRNA than BM-derived DC from the diabetes-resistant strain, B10.BR. We have also investigated the ability of these DC populations to modulate the development and progression of diabetes in NOD mice.     

Role of the first external domain of I-A beta chain in immune responses and diabetes in non-obese diabetic (NOD) mice.

Diabetes in the non-obese diabetic (NOD) mouse is a multigenic autoimmune disease and is possibly controlled by three recessive loci, including one that is linked to the major histocompatibility complex (MHC). The first external domain of the Class II MHC I-A beta chain in these mice is unique and has been suggested as being responsible for autoimmunity. The I-A alpha chain in these mice is I-A alpha d, and they lack the expression of I-E molecules. We have investigated immune responses to various Ir gene control antigens in NOD mice to determine the influence of the NOD Ia and particularly the I-A beta chain. We find that sheep insulin is highly immunogenic while other insulins are weakly immunogenic in these mice. Hen egg lysozyme, pigeon cytochrome C and the synthetic polypeptide Poly 18, Poly EYK(EYA)5 antigen produce good antibody responses. Apart from H-2d, NOD are the only mice where Poly 18 antigen is immunogenic. In these mice Poly 18 induced good T-cell proliferative response, which was inhibited by anti-Ia antibody, and the mice were able to respond to tyrosine-containing polypeptide Poly EYA but not to the phenylalanine-containing antigen Poly EFA. We also found that synthetic peptide 48-60 of the NOD I-A beta chain is highly immunogenic in syngeneic NOD mice both for T cells and B cells. Using an I-A beta chain-specific monoclonal antibody, we are able to prevent induction of diabetes when the antibody was administrated in prediabetic, young mice. Our results suggest that the immune response to various antigens and autoimmune diabetes in NOD mice is directly influenced by the I-A beta chain.     

Transient insulin autoantibody expression independent of development of diabetes: comparison of NOD and NOR strains

NOD mice spontaneously develop anti-insulin autoantibodies associated with the subsequent development of diabetes. NOD mice that express insulin autoantibodies at 8 weeks of age have a diabetes risk exceeding 90%, while mice that do not express autoantibodies by 16 weeks have a risk of less than 20%. NOD female mice expressed insulin autoantibodies more often than male mice (13/15+ vs. 6/15+). Autoantibodies characteristically developed between 8 and 20 weeks and then for most mice became negative at diabetes onset in NOD mice. In the diabetes-free strain NOR mice, spontaneous expression of insulin autoantibodies was observed in less mice (female 8/15+, male 3/10+) compared to NOD mice. The expression of autoantibodies was transient in NOR mice and followed the same time-course as for NOD mice and they were all negative by 28 weeks (without progression to diabetes). No correlation was found in NOR mice between the levels of autoantibodies and insulitis. The program of insulin autoantibody expression is regulated over approximately 5 months for both NOD and NOR mice with only NOD mice developing diabetes, indicating that depending upon genetic combination, the presence of insulin autoantibodies does not always predict diabetes development. In addition, this data is not consistent with the hypothesis that the time-course of autoantibodies simply reflects the destruction of beta-cells with development of diabetes.     

Induction of diabetes with islet-specific T-cell clones is age dependent.

To investigate the effects of recipient age on the induction of diabetes by CD4+ islet-specific T-cell clones, we tested four different clones in non-obese diabetic (NOD) mice of different ages. Transfer of each of the T-cell clones resulted in insulitis and full development of diabetes in unirradiated 8-18-day-old NOD mice within 2-3 weeks. A small gender bias in disease susceptibility was seen in 8-14-day-old female NOD mice, which showed a twofold increase in disease incidence over both age-matched males and slightly older (15-18-day-old) females. In contrast, both male and female NOD mice, 19 days or older, were highly resistant to T-cell clone-induced insulitis and completely resistant to the development of diabetes. In mice of an intermediate age range (15-18 days old), two of the clones showed a three- to fourfold reduction in transfer of overt diabetes regardless of gender. These results suggest that an important developmental event occurs in both male and female NOD mice between the age of 15 and 19 days, leading to the inhibition of disease induced by T-cell clones.