Effects of Irradiation on Diabetes in the BB/WOR Rat

Lymphoid irradiation is known to prevent spontaneous autoimmune diabetes in susceptible BB rats. The present studies investigated further the effects of radiation in diabetes prone (DP) and resistant (DR) BB/Wor rats, and histocompatible Yoshida (YOS) rats. Single doses of total body gamma irradiation (125–600 rads) induced diabetes within 22–44 days in 20 of 102 (20%) 30 day old DR rats, < 1% of which develop the disease. Radiation was also associated with (1) a reduction in the ratio of W3/25⁺ to OX8⁺ peripheral blood lymphocytes within 2 weeks, and (2) a decreased percentage of lymph node cells expressing the RT6.1 surface alloantigen 3–4 weeks after treatment. Similar doses of irradiation did not alter the frequency or age at onset of diabetes in DP rats, and did not induce diabetes in YOS rats. When a single dose of 250 or 500 rads of gamma irradiation was followed by injection of mitogen activated spleen cells from acutely diabetic rats to adoptively transfer diabetes, 16 of 19 (84%) DR and 8 of 14 (57%) YOS rats became diabetic. Long term exposure to ultraviolet irradiation (UVB) did not alter the frequency or age at onset of diabetes in either DP or DR rats. We conclude that there may exist a population of regulatory cells relatively sensitive to gamma irradiation that play a role in determining the susceptibility of rats to autoimmune diabetes mellitus.     

Anti-CD2 monoclonal antibodies prevent spontaneous and adoptive transfer of diabetes in the BB/Wor rat.

We studied the effects of anti-CD2 monoclonal antibodies (MAb) on spontaneous and induced autoimmune diabetes mellitus in diabetes-prone (DP) and diabetes-resistant (DR) BB/Wor rats. In DP rats, all anti-CD2 MAb prevented spontaneous diabetes and the adoptive transfer of diabetes with Con-A--stimulated acute diabetic spleen cells; OX34 prevented Poly I:C induced accelerated onset of diabetes and the adoptive transfer of diabetes with Con-A--stimulated RT6.1+ T cell depleted DR splenocytes. In DP rats, all anti-CD2 MAb except OX53 depleted CD4+ T cells, without depleting natural killer cells or CD8+ T cells. OX34 injected DR rats were profoundly depleted of CD4+ T cells without evidence of decreased CD8+ T cells, but were not protected against the induction of diabetes by RT6.1+ T-cell depletion and Poly I:C injections. We conclude that anti-CD2 MAbs protect against BB/Wor autoimmune diabetes by depleting CD4+ T cells, preventing the activation of effector cells, or by blocking CD2/ligand interaction between effector and target cells.     

Quantitative analyses comparing all major spleen cell phenotypes in BB and normal rats: autoimmune imbalance and double negative T cells associated with resistant, prone and diabetic animals.

The BB rat is a model of spontaneous autoimmune diabetes. To characterize quantitatively all known immune cell subsets involved in disease pathogenesis, FACS analysis of spleen cells was performed in diabetes-prone (DP) and acutely diabetic (D) BB rats and compared with diabetes-resistant (DR) BB and normal Wistar-Furth (WF) strains. We observed increased percentages of splenic NK cells in DP and D animals compared with DR rats using an NK-specific monoclonal antibody. We found increased proportions of splenic macrophages in the T-lymphopenic DP and D rats and low macrophage contents in DR spleens compared with WF spleens. We observed that percentages of the CD4-CD8- T cell receptor alpha/beta+ (double-negative) T cell subset were strikingly increased in the lymphopenic DP and D animals, compared with DR animals. We observed increased percentages of activated splenic CD5+ T cells expressing the IL-2 receptor and MHC class II antigen in DP and D rats compared with DR animals. Our studies suggest that (a) splenic NK cells and macrophages quantitatively appear to be involved in the pathogenesis of diabetes; (b) double-negative T cells escape from the T cell depletion process; (c) a marked increase of activated splenic T cells suggests diabetes is associated with general T cell activation processes; and (d) an altered balance among the different immune cell subsets may in part explain the pathogenesis of diabetes, since marked relative changes are observed when comparing the DR strain to the DP strain in both the prediabetic and diabetic stages.     

Depletion of RT6.1+ T lymphocytes alone is insufficient to induce diabetes in diabetes-resistant BB/Wor rats.

Diabetes-prone (DP) BB/Wor rats are lymphopenic, lack RT6.1+ T cells, and spontaneously develop diabetes mellitus. Diabetes-resistant (DR) rats are not lymphopenic, have normal numbers of RT6.1+ T cells, and rarely become diabetic. It has been reported that RT6.1+ T-cell depletion induced insulitis and diabetes in DR rats. To study the effector cells responsible for diabetes in DR rats after RT6+ T cell depletion, we treated a large number of intact and 21-day thymectomized DR rats with anti-RT6.1 monoclonal antibody, anticipating a high frequency of insulitis and diabetes. Our treatment protocol depleted RT6+ T cells but failed to induce insulitis and diabetes with the expected frequency in either intact or thymectomized DR animals. After Con-A stimulation however, RT6-depleted DR spleen cells readily transferred diabetes to DP recipients. These results suggested that while RT6+ T-cell depletion alone was permissive for the induction of diabetes in BB/Wor DR rats, a second stimulus was required to activate the DR effector cells responsible for insulitis and diabetes. In view of the occasional presence of pancreatitis and peritonitis in the diabetic RT6.1+ T-cell-depleted DR rats, additional experiments were performed in which RT6-depleted DR rats were also given intraperitoneal injections of sterile fecal suspensions to deliberately induce peritoneal inflammation. Insulitis and diabetes were significantly increased in these rats, lending credence to the requirement of a second (environmental) stimulus for the induction of diabetes in RT6-depleted BB/Wor DR rats.     

Significant glomerular basement membrane thickening in hyperglycemic and normoglycemic diabetic-prone BB Wistar rats

The diabetic-prone BioBreeding Wistar rat (BB/DP) is an autoimmune model of insulin-dependent diabetes mellitus. Approximately 80-90% of the animals are hyperglycemic (BB/DP(h)) by 90-120 days of age while those that do not become diabetic in adolescence (BB/DP(n)) remain normoglycemic for life. Likewise, rats in the diabetes-resistant (BB/DR) strain are normoglycemic. Although renal morphological studies have been carried out in this model, ultrastructural observations of age- and diabetes-related extracellular matrix (ECM) changes, including glomerular basement membrane (GBM) morphometry, are not available. Moreover, possible renal changes in the relatively uncommon BB/DP(n) control animals have not been reported. The current electron microscopic study was carried out to investigate temporal changes in detergent-treated acellular ECM in BB/DP(h) rats at 2 weeks, 3 months, 6 months, and 1 year postonset of moderate hyperglycemia. Age-matched BB/DR and BB/DP(n) control animals were also examined. Our data demonstrate age- and diabetes-related alterations in mesangial matrix distributions and GBM widths and show for the first time significant increases in GBM thickening in both hyperglycemic (BB/DP(h)) and normoglycemic (BB/DP(n)) rats when compared to age-matched BB/DR controls. Surprisingly, the rate of increase is greatest in BB/DP(n) animals. Although the pathogenesis of diabetic basement membrane disease is not completely understood, GBM thickening is widely regarded as a morphological consequence of hyperglycemia. However, data in the current investigation show that ECM alterations, including significantly increased GBM thickness, may occur in genetically diabetic animals in the absence of hyperglycemia.     

In vivo effects of interleukin-1 beta on blood leukocytes in BB rats prone or resistant to diabetes.

Previous studies have determined that daily low dose injections of the potent cytokine interleukin-1 beta (IL-1 beta) decreased the frequency of insulin-dependent diabetes mellitus (IDDM) in diabetes-prone (DP) BB rats. In contrast, high dose injections induced an earlier than normal onset. In this study we tested whether the effects of daily human recombinant IL-1 beta injections on leukocyte subsets were associated with its modulation of IDDM onset in BB rats. Prior to the onset of IDDM in DP BB rats, high dose IL-1 beta induced leukocytosis (P less than 0.05), neutrophilia (P less than 0.01), and monocytosis (P less than 0.001). At the onset of IDDM, lymphocyte (P less than 0.01) and neutrophil (P less than 0.001) numbers were increased in high dose treated DP rats but not in rats given saline or low dose IL-1 beta. In 60-day-old diabetes-resistant (DR) BB rats, neurophilia was induced by both low (P less than 0.05) and high (P less than 0.001) dose IL-1 beta without the development of IDDM. At 130 days of age, when the rats were killed, it was discovered that 14/22 (64%) IL-1 beta injected DR rats developed neutralizing IL-1 beta antibodies. Significantly lower neutrophil numbers were observed in high dose DR rats which developed IL-1 beta antibodies compared with those which did not (P = 0.032). Thus, neutrophilia was dissociated from high IL-1 beta acceleration of IDDM onset.(ABSTRACT TRUNCATED AT 250 WORDS)     

Significant retinal capillary basement membrane thickening in hyperglycemic and normoglycemic diabetic-prone (DP) BB Wistar rats

The diabetic-prone BioBreeding Wistar (BB/DP) rat is an autoimmune model of insulin-dependent diabetes mellitus. Approximately 90% of the animals (BB/DPh) are hyperglycemic by 90-120 days of age, while the remaining ~10% (BB/DPn) and diabetes-resistant rats (BB/DR) are normoglycemic for life. The transmission electron microscope data from this study demonstrate expected significant age- and diabetes-related increases in retinal capillary basement membrane (RCBM) widths in (BB/DPh) rats relative to BB/DR animals. However, the data show, for the first time, an unexpected significant RCBM thickening in (BB/DPn) rats compared to BB/DR animals at 6 months and 1 year post-onset of hyperglycemia.     

Thymic epithelial defects and predisposition to autoimmune disease in BB rats.

We report an association between thymic epithelial defects and predisposition to autoimmunity. Diabetes-prone (DP) BB rats develop spontaneous hyperglycemia and are deficient in T cell subsets expressing the RT6 alloantigen. Diabetes resistant (DR) BB rats become diabetic if depleted of RT6+ T cells. The inciting immune system defects are unknown. We made the following observations: 1) Regions of thymic cortex and medulla devoid of thymic epithelium exist in DP-BB, DR-BB, and Lewis rats, all of which are susceptible to autoimmune disorders. Such defects were absent in eight normal rat strains. 2) Thymic epithelial defects are absent at birth, but present in BB rats at 4 weeks of age. 3) The genetic predisposition to thymic epithelial defects is an autosomal dominant trait. 4) The observation of thymic defects in (DP x WF)F1 rats led to the prediction that such animals, which never develop spontaneous autoimmunity, might be susceptible to its induction. Following depletion of RT6+ T cells we observed diabetes in 91%, and thyroiditis in 43%, of treated F1 animals (n = 23). Pancreatic insulitis was uniformly present. Because thymic epithelium participates in the positive and negative selection of developing thymocytes, we propose that thymic epithelial defects may play an important role in the predisposition of BB rats to autoimmunity.     

Defects in the thymic epithelial stroma of diabetes prone BB rats

The thymus of diabetes prone BB rats (DP) was studied and compared with diabetes resistant (DR) BB rats and normal WAG rats. Thymuses were obtained from 4-5 week old animals, i.e. before the onset of disease. Analysis included specific immune histology using a panel of monoclonal antibodies directed against markers both on thymocytes and stromal cells. No striking differences were observed between the three groups with regard to the expression and distribution of the various T cell markers. There was however a marked difference for thymic class II MHC antigen expression between the various groups. Whereas WAG rats displayed a regular class II MHC pattern both in the cortex and the medulla, both DR and DP rats showed large areas in the cortex where there was no class II MHC staining. The lack of expression of class II MHC antigens in these 'holes' was associated with a complete absence of epithelial cells in that area. Also in the medulla of DP and DR thymuses 'holes' in the keratin-stroma were observed. The stromal aberrations in these auto-immune prone rats are discussed in the context of the deficient T cell system in these animals.     

DR.lyp/lyp bone marrow maintains lymphopenia and promotes diabetes in lyp/lyp but not in +/+ recipient DR.lyp BB rats

Lymphopenia is due to a frameshift mutation in Gimap5 on rat chromosome 4 and is linked to type 1 diabetes in the diabetes prone (DP) BB rat. The hypothesis that bone marrow derived cells confer the lymphopenia phenotype was tested by reciprocal bone marrow transplantation in 40-day-old lethally irradiated diabetes resistant (DR) congenic DR.lyp/lyp (lymphopenia and diabetes) and DR.+/+ (no lymphopenia and no diabetes) rats. In two independent series of transplants, all DR.lyp/lyp rats (n=5 and 4) receiving DR.lyp/lyp bone marrow retained lymphopenia and developed insulitis (5/5 and 4/4) as well as diabetes in some (2/5 and 3/4). Both DR.+/+ and DR.lyp/lyp rats receiving DR.+/+ bone marrow cells as well as DR.+/+ rats receiving DR.lyp/lyp bone marrow cells showed no lymphopenia or diabetes. In accordance with earlier studies in non-congenic BB rats, the DR.+/+ rats receiving DR.lyp/lyp bone marrow cells recapitulated an intermediary phenotype rather than the +/+ or lyp/lyp phenotypes. Our data demonstrate that BBDP rat lymphopenia and diabetes are transferred by bone marrow transplantation to syngeneic DR.lyp/lyp but not DR.+/+ recipients. The intermediary recapitulation of DR.lyp/lyp T cells in recipient DR.+/-/+/- rats suggests that radiation resistant +/-/+/- T cells, the Gimap5 mutation in bone marrow cells, or both may not support the development of lymphopenia.     

Defects in differentiation of bone-marrow derived dendritic cells of the BB rat are partly associated with IDDM2 (the lyp gene) and partly associated with other genes in the BB rat background

BB rats develop various organ-specific autoimmune diseases, e.g. autoimmune diabetes and thyroiditis and have proven important to dissect genetic factors that govern autoimmune disease development. The lymphopenia (lyp) gene (iddm2) is linked to autoimmune disease development and is a major genetic difference between diabetes-resistant (DR) and diabetes-prone (DP) BB rats. To study the effects of the lyp gene and other genes on dendritic cell (DC) differentiation from bone-marrow precursors, such differentiation was studied in BB-DP, BB-DR, Wistar and F344 control rats. DC of BB-DP rats showed a lower MHC class II expression as compared to BB-DR, Wistar and F344 rats. LPS-maturation did not restore this low MHC class II expression. DC of BB-DP rats also showed a poor capability to terminally differentiate into mature T cell stimulatory DC under the influence of LPS and produced significantly lower quantities of IL-10, yet these aberrancies were also found in BB-DR rats but did not occur in control rats. This study thus shows that various aberrancies exist in the differentiation of myeloid DC from bone-marrow precursors in the BB rat model of organ-specific autoimmunity. These aberrancies are multigenically determined and partly associated with iddm2 (lyp gene) and partly associated with other genes in the BB rat.     

High Dose Nicotinamide Fails to Prevent Diabetes in BB Rats

Nicotinamide which is an inhibitor of poly (ADPR) synthetase and precursor of NAD has been observed to prevent diabetes in some experimental models possibly by protecting beta cells. To determine whether nicotinamide could cure or prevent type 1 diabetes, we administered large doses (0.5 g/Kg/d) to BB rats. When used in the 45 days following diagnosis nicotinamide failed to bring remission. As a preventive treatment, nicotinamide administered between the 40th and 90th day of age, alone or in association with desferrioxamine did not significantly lower the incidence of diabetes (23% and 30.8% respectively vs. 56.6%). When used earlier, immediately after weaning, nicotinamide did not affect the incidence of diabetes in this model (62.5%). The degree of protection was not comparable with that obtained with cyclosporin A (15% of diabetic animals). Histology study of the pancreas from the animals killed either immediately or 1 year after treatment revealed no endocrine tumor. These findings suggest that in BB rats nicotinamide has little or no effect on the course of autoimmune diabetes mellitus thus dampening the high hopes for this drug in the treatment of human diabetes.     

High dose nicotinamide fails to prevent diabetes in BB rats

Nicotinamide which is an inhibitor of poly (ADPR) synthetase and precursor of NAD has been observed to prevent diabetes in some experimental models possibly by protecting beta cells. To determine whether nicotinamide could cure or prevent type 1 diabetes, we administered large doses (0.5 g/Kg/d) to BB rats. When used in the 45 days following diagnosis nicotinamide failed to bring remission. As a preventive treatment, nicotinamide administered between the 40th and 90th day of age, alone or in association with desferrioxamine did not significantly lower the incidence of diabetes (23% and 30.8% respectively vs. 56.6%). When used earlier, immediately after weaning, nicotinamide did not affect the incidence of diabetes in this model (62.5%). The degree of protection was not comparable with that obtained with cyclosporin A (15% of diabetic animals). Histology study of the pancreas from the animals killed either immediately or 1 year after treatment revealed no endocrine tumor. These findings suggest that in BB rats nicotinamide has little or no effect on the course of autoimmune diabetes mellitus thus dampening the high hopes for this drug in the treatment of human diabetes.     

Mechanisms of complete Freund's adjuvant protection against diabetes in BB rats: induction of non-specific suppressor cells.

We have previously reported that a single injection of complete Freund's adjuvant (CFA) can prevent diabetes appearance in diabetes-prone (DP) BB rats. In this study, we investigated further the mechanism of CFA-induced protection from diabetes. We found that adoptive transfer of splenic cells from CFA-treated DP rats into young DP rats protected the latter from diabetes development. This suggested that CFA-induced protection from diabetes resulted from activation of regulatory (suppressor) cells. Cell mixing experiments in vitro indicated that CFA activated splenic cells with antigen-nonspecific suppressor activity (suppression of lymphoproliferative responses to lipopolysaccharide and to allogeneic splenic cells). Fractionation of splenic cells on Percoll revealed that the suppressor activity resided in low density cells relatively depleted of T-cells, B-cells, macrophages and NK cells. These results suggest that non-specific (natural) suppressor cells in CFA-treated BB rats may be responsible for suppressing autoimmune responses and preventing insulitis and diabetes development.     

Prediction of diabetes in Biobreeding/Aburahi rats by the measurement of soluble L-selectin

L-selectin was initially identified as a homing receptor, recently soluble L-selectin has been used as a marker of the inflammation. Therefore, we investigated the relation between the development of diabetes and serum L-selectin levels in the Biobreeding (BB) rats. Serum L-selectin were measured from 30 days old to the onset of diabetes or to 90 days old in Biobreeding (BB) rats and Wistar Furth (WF) rats. Significant elevation of L-selectin was found in diabetes prone (DP) rats from 45 days old to the onset of diabetes or through 90 days old. No elevation was found in other strain of rats. In histological study, all of DP rats had insulitis and no other strain of rats had it. Therefore, we conclude that the measurement of serum L-selectin could be useful tool to predict the onset of diabetes or presence of insulitis in BB rats.     

Alleles of diabetes-resistant BN rats contribute to insulin-dependent type 1 diabetes mellitus

Diabetes in the biobreeding (BB) rat results from autoimmune destruction of pancreatic beta cells and thereby it is sharing many features with human type 1 diabetes. Independent crossing studies have demonstrated that diabetes in the BB rat is explained by at least three recessively acting genes termed Iddm1 (major histocompatibility complex), Iddm2 (lymphopenia), Iddm3 (unknown). About 50% of Iddm1 and Iddm2 homozygous first backcross hybrids (BC1) usually develop diabetes. However, 75% of these homozygotes become diabetic when using diabetic BB/HRI and diabetes-resistant BN/Mol rats. That prompted us to carry out a cross between BB/OK and BN/Crl rats in order to localise diabetogenic gene(s) of BB and/or BN rats.Fifty nine Iddm1 and Iddm2 homozygous [(BNxBB)F1xBB] BC1 hybrids (35 M, 24 F) were observed for diabetes occurrence up to an age of 30 weeks. All hybrids were used in a genome-wide scan carried out with 238 microsatellite markers covering about 92% of the genome.Significantly more Iddm1 and Iddm2 homozygous BC1 hybrids became diabetic (69 vs. 50%, p<0.003) with an age at onset of 91+/-31 days. Significant deviations from expected allele distribution between diabetic and non-diabetic BC1 hybrids were found at loci on chromosomes 1, 2, 3, 9, 10, 15, 16 and 19, with the strongest effect observed at locus D10Mgh2, where more heterozygous (91%) than homozygous diabetics (44%) were found. We conclude that BN rats possess more than one gene contributing to type 1 diabetes development.     

Prediction of Diabetes in Biobreeding/Aburahi Rats by the Measurement of Soluble L-Selectin

L-selectin was initially identified as a homing receptor, recently soluble L-selectin has been used as a marker of the inflammation. Therefore, we investigated the relation between the development of diabetes and serum L-selectin levels in the Biobreeding (BB) rats. Serum L-selectin were measured from 30 days old to the onset of diabetes or to 90 days old in Biobreeding (BB) rats and Wistar Furth (WF) rats. Significant elevation of L-selectin was found in diabetes prone (DP) rats from 45 days old to the onset of diabetes or through 90 days old. No elevation was found in other strain of rats. In histological study, all of DP rats had insulitis and no other strain of rats had it. Therefore, we conclude that the measurement of serum L-selectin could be useful tool to predict the onset of diabetes or presence of insulitis in BB rats     

Diabetes prone BB rats are severely deficient in natural killer T cells

Diabetes prone (DP) BB rats develop spontaneous autoimmune hyperglycemia. Coisogenic diabetes resistant (DR) BB rats develop diabetes in response to immunological and environmental perturbants, but not spontaneously. Both are used to model human insulin-dependent diabetes mellitus (IDDM). Deficiencies in natural killer (NK) T cells have been implicated in the expression of human IDDM, but little is known of their phenotype or function in the rat. We now report that the phenotype of NK T cells in the rat is alphabetaTcR+ CD8+ CD4-, comparable to the NK T cell phenotype reported for humans, which is alphabetaTcR+ CD4- Valpha24-JalphaQ, and either CD8- or CD8alphaalpha+. We also report that DP- but not DR-BB rats are severely deficient in splenic and intrahepatic NKR-P1+ alphabetaTcR+ (NK T) cells. Because RT6+ T cells are deficient in DP-BB rats, and because depletion of cells expressing RT6 induces IDDM in DR-BB rats, we studied NK T cells for expression of this antigen. We observed that the majority of rat NK T cells express RT6+. In addition, injection of cytotoxic anti-RT6.1 monoclonal antibody depleted splenic and intrahepatic RT6+ NK T cells, T cells, and NK cells, but left intact the RT6- subset of each population. These results suggest that deficiencies in NK T cells may play a role in the susceptibility of DP- and DR-BB rats, respectively, to spontaneous and induced autoimmune IDDM.     

A pancreatic venular defect in the BB/Wor rat.

BB rats develop spontaneous autoimmune diabetes mellitus characterized morphologically by insulitis, an inflammatory lymphocytic infiltration of the islets of Langerhans. To investigate the role of the vascular endothelium of the pancreas in this destructive process, the authors injected diabetes-prone (DP) and diabetes-resistant (DR) BB/Wor rats as well as other nondiabetic strains of rats with Monastral blue B, a colloidal pigment that identifies leaky microvasculature. They found evidence of a venular defect limited to the pancreas that is specific to the BB rat. Light- and electron-microscopic evidence suggests that this defect is due to a population of trapped (marginating) intravascular monocytes, which may be activated by the colloidal pigment and release vasoactive mediators.     

Prevention of diabetes in the BB rat by early immunotherapy using Freund's adjuvant

The BB rat spontaneously develops an insulin-dependent diabetes mellitus (IDDM) that closely resembles this disease in man. The pathogenesis involves autoimmune destruction of pancreatic islet beta-cells. In the present study, a single intraperitoneal injection of complete Freund's adjuvant (CFA) in diabetes-prone (DP) BB/Wor rats between 9 and 28 days of age reduced the incidence of diabetes at 120 days from 89% to 10-28%, whereas injection of CFA after 40 days of age was ineffective. The CFA-injected, diabetes-free DP rats had normal levels of pancreatic insulin and little or no mononuclear leukocyte infiltration in the islets. These protective effects of CFA were not associated with any changes in peripheral blood leukocyte counts or monoclonal antibody-defined T cell, B cell, or macrophage subsets in the spleen of the DP rats. These results suggest that it is possible to prevent diabetes by adjuvant treatment in early life without general immunosuppression or sustained therapy.