Insulin immunization of nonobese diabetic mice induces a protective insulitis characterized by diminished intraislet interferon-gamma transcription.

We reported previously that daily injections of isophane insulin prevented both hyperglycemia and insulitis in nonobese diabetic (NOD) mice (Atkinson, M., N. Maclaren; and R. Luchetta. 1990. Diabetes. 39:933-937). The possible mechanisms responsible include reduced immunogenicity of pancreatic beta-cells from "beta-cell rest" and induced active immunoregulation to insulin (Aaen, IK., J. Rygaard, K. Josefsen, H. Petersen, C. H. Brogren, T. Horn, and K. Buschard. 1990. Diabetes. 39:697-701). We report here that intermittent immunizations with insulin or its metabolically inactive B-chain in incomplete Freund's adjuvant also prevent diabetes in NOD mice, whereas immunizations with A-chain insulin or with BSA do not. Adoptive transfer of splenocytes from B-chain insulin-immunized mice prevented diabetes in recipients co-infused with diabetogenic spleen cells, an effect that was abolished by prior in vivo elimination of either CD4+ or CD8+ cells. Insulin immunization did not reduce the extent of intraislet inflammation (insulitis); however, it did abolish expression of IFN-gamma mRNA within the insulitis lesions. Immunizations with insulin thus induce an active suppressive response to determinants on the B-chain that converts the insulitis lesion from one that is destructive to one that is protective.

Insulitis and diabetes in NOD mice reduced by prophylactic insulin therapy.

Intensive insulin therapy in patients with recently diagnosed insulin-dependent diabetes mellitus (IDDM) has been reported to result in a prolonged increase in endogenous insulin-secreting capacity. Because the clinical onset of IDDM occurs only after most insulin-secreting beta-cells have been destroyed, we tested whether prophylactic insulin therapy might prevent IDDM in nonobese diabetic (NOD) mice. One hundred fourteen NOD mice were randomized at weaning into a protamine zinc pork insulin-treated (I) group or a placebo-treated (P) group given insulin diluent. All insulin treatments were adjusted to the maximum tolerable dosages and continued until 180 days of age. The cumulative IDDM frequency within the female I group was significantly less (3 of 34, 8%) than in female P controls (17 of 26, 65%; P less than 0.0001). This beneficial effect was limited to females, however, because the frequency of IDDM in male I mice (3 of 32, 9%) was not significantly different from the frequency in male P controls (1 of 22, 5%; P less than 0.5). Pancreatic histological examinations of nondiabetic animals revealed that insulin treatment resulted in significant reductions in islet cell inflammation and damage and improvements in insulin content. In summary, NOD mice given insulin therapy from weaning until 180 days of age had significantly lower frequencies of diabetes and pancreatic insulitis than sex-matched control littermates treated with insulin diluent. These results suggest that prophylactic insulin therapy to prevent IDDM in humans should be considered for clinical trials.

Immunogenetic analysis of beta-cell autoimmunity in NOD mice. Relationship of insulitis to T-lymphocyte-receptor beta nod and A beta nod genes.

An early molecular event in the evolution of insulin-dependent diabetes in humans and NOD mice appears to involve the interaction of MHC class II molecules, beta-cell autoantigen-derived peptides, and receptor molecules of helper T lymphocytes. To examine the influence of T-lymphocyte-receptor beta-genes on the development of beta-cell autoimmunity, (NOD x NZW)F1 x NOD backcrossed (BC) mice were studied for the development of insulitis, because insulitis is the pathognomonic histological lesion of autoimmune diabetes. Heterozygosity for H-2nod was permissive for the development of pancreatic interstitial inflammation and peri-islet insulitis, whereas homozygosity for H-2nod was highly associated with insulitis. However, (NOD x NZW)F1 x NOD BC mice developed insulitis regardless of homozygosity or heterozygosity for T-lymphocyte receptor beta nod. Therefore, in our study, T-lymphocyte receptor beta nod did not function as an autosomal-recessive beta-cell autoimmunity gene.

Pertussigen treatment retards, but fails to prevent, the development of type I, insulin-dependent diabetes mellitus (IDDM) in NOD mice.

Current evidence supports an autoimmune etiopathogenesis for Type I, insulin-dependent diabetes mellitus (IDDM) in which the pancreatic beta (beta) cell is the specific target tissue. Recently, the NOD (non-obese diabetic) mouse has become an important model for IDDM, exhibiting many of the pathological features observed in man, including a progressive pancreatic islet leukocytic inflammation referred to as insulitis. The present study was carried out to determine the efficacy of the bacterial-derived bio-product, pertussigen, to retard the progression of insulitis and thereby prevent overt diabetes. Results revealed that (1) the rapid onset of IDDM in female NOD mice is absent if the mothers are treated with pertussigen prior to mating, (2) treatment of young prediabetic NOD mice with repeated injections of pertussigen results in the retardation of onset of IDDM when compared to untreated control NOD mice, and (3) the severity of insulitis in pertussigen-treated NOD mice not developing IDDM was noticeably less severe than age and sex-matched untreated control mice. Since earlier work had shown that pertussis vaccine, which contains pertussigen, could prevent development of IDDM in mice treated with streptozotocin, the present results may indicate basic differences in the inflammatory responses in the genetically-predisposed NOD mice and IDDM-nonsusceptible mice with streptozotocin-induced diabetes.

T cell receptor beta diversity and joining segments in the NOD mouse.

Pancreatic beta-cell autoantigen recognition by the immune system appears to be a critical event in the evolution of insulin dependent diabetes. Immune recognition involves antigen presentation by macrophages and subsequent antigen-peptide-class II MHC recognition by T cell receptors (TCR). Using the NOD mouse as a model for human IDD, we hypothesized that germline variability in the D beta nod and/or J beta nod segments could contribute to beta cell autoimmunity by influencing the specific peptides that are recognized. As an initial approach to our hypothesis, we sought to compare these segments to other strains of mice in search of genetic polymorphisms as reported in NZW mice. The germ line TCR beta nod gene did not display evidence of an expansion or contraction in the number of D beta nod or J beta nod segments at the level of resolution provided by restriction fragment length polymorphism analysis. The absence of such polymorphisms suggests that D beta nod or J beta nod segments are not different from nonautoimmune strains of mice.

Pharmacokinetics of aminoguanidine administration and effects on the diabetes frequency in nonobese diabetic mice.

In vitro studies suggest that intra-islet nitric oxide production may contribute to the pathogenesis of autoimmune insulin-dependent diabetes mellitus. We tested whether aminoguanidine (AG), a competitive inhibitor of inducible nitric oxide synthase, might block beta cell destruction and prevent insulin-dependent diabetes mellitus in vivo. A total of 50 female nonobese diabetic mice, from the time of weaning until 32 wk of age, received injections (i.p.) twice daily with 50 mg AG/kg body weight and received AG in drinking water (350 mg/liter). A total of 50 littermates treated with vehicle alone served as controls. A 24-hr pharmacokinetic analysis showed that AG was readily absorbed after i.p. administration, peaked in plasma (9.0 micrograms/ml) at 0.5 hr and had a half-life of 1.88 hr. Steady-state values for the area under the curve for the therapeutic regimen were 20.51 and 16.35 (micrograms)(hr)/ml for the 0000 to 1600 and 1600 to 2400 hr, respectively. In terms of therapy, life-table analysis indicated the frequency of insulin-dependent diabetes mellitus (6/30 AG-treated vs. 11/31 vehicle-treated, P = .25) and insulitis scores (2.0 +/- 1.1 vs. 2.4 +/- 1.2 in nondiabetic AG- and vehicle-treated mice at 32 wk, respectively, P = .20) were similar in both groups. Flow cytometric analysis revealed no quantitative differences in islet infiltrating macrophages, CD4+ or CD8+ T lymphocytes between groups of animals randomly killed at 8, 16 and 32 wk. Although not eliminating a role for nitric oxide in the pathogenesis of insulin-dependent diabetes mellitus, prophylactic treatment with AG did not significantly impact the onset of insulitis or diabetes in nonobese diabetic mice.