BioBreeding Rats Research Articles

Genetic isolation of iddm 1 on chromosome 4 in the biobreeding (BB) rat.

Genetic isolation of iddm 1 on chromosome 4 in the biobreeding (BB) rat.

Cellular and Molecular Mechanisms for the Initiation and Progression of β Cell Destruction Resulting from the Collaboration Between Macrophages and T Cells

Insulin-dependent diabetes mellitus (IDDM) is caused by the progressive autoimmune destruction of insulin-producing pancreatic β cells. Although the pathogenesis of autoimmune IDDM has been extensively studied, the precise mechanisms involved in the initiation and progression of β cell destruction remain unclear. Animal models used in the study of IDDM, such as the BioBreeding (BB) rat and the nonobese diabetic (NOD) mouse, have greatly enhanced our understanding of the pathogenic mechanisms involved in this disease. In these animals, macrophages and/or dendritic cells are the first cell types to infiltrate the pancreatic islets. Macrophages must be involved in the pathogenesis of IDDM early on, since inactivation of macrophages results in the near-complete prevention of insulitis and diabetes in both NOD mice and BB rats. The presentation of β cell-specific autoantigens by macrophages and/or dendritic cells to CD4+ T helper cells, in association with MHC class II molecules, is considered the initial step in the development of autoimmune IDDM. The activated macrophages secrete IL-12, which stimulates Thl type CD4+ T cells. The CD4+ T cells secrete IFN-γ and IL-2. IFN-γ activates other resting macrophages, which, in turn, release cytokines, such as 1L-1 β, TNF-α, and free radicals, which are toxic to β cells. During this process, IL-2 and other cytokines induce the migration of CD8+ peripheral T cells to the inflamed islets, perhaps by inducing the expression of a specific homing receptor. The precytotoxic CD8+ T cells that bear β cell-specific autoantigen receptors differentiate into cytotoxic effector T cells upon recognition of the β cell-specific peptide bound to MHC class I molecules in the presence of β cell-specific CD4+ T helper cells. The cytotoxic CD8+ T cells then effect β cell damage by releasing perforin and granzyme, and by Fas-mediated apoptosis. In this way, macrophages, CD4+ T cells, and CD8+ T cells synergistically destroy β cells, resulting in the onset of autoimmune IDDM.

Antigen-presenting dendritic cells as regulators of the growth of thyrocytes: a role of interleukin-1beta and interleukin-6.

An accumulation of antigen-presenting dendritic cells (DC) in the thyroid gland, followed by thyroid autoimmune reactivity, occurs in normal Wistar rats during iodine deficiency, and spontaneously in diabetic-prone Biobreeding rats. This intrathyroidal DC accumulation coincides with an enhanced growth rate and metabolism of the thyrocytes, suggesting that both phenomena are related. Because DC are known to regulate the hormone synthesis and growth in other endocrine systems (i.e. the pituitary, the ovary, and the testis), we tested the hypothesis that DC, known for their superb accessory cell function in T cell stimulation, act as regulators of thyrocyte proliferation (and hormone secretion). We investigated the effect of (Nycodenz density gradient) purified splenic DC from Wistar rats on the growth rate of and thyroid hormone secretion by Wistar thyroid follicles (collagenase dispersion) in culture. Various numbers of DC and follicles were cocultured during 24 h. The proliferative capacity of thyrocytes was measured by adding tritiated thymidine (3H-TdR) and bromodeoxyuridine, the hormone secretion into the culture fluid was measured by using a conventional T3 RIA. Furthermore, antibodies directed against interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) were added to these cocultures to determine the role of these cytokines in a possible DC regulation of thyrocyte growth. Cocultures were also carried out in the presence of antimajor histocompatibility complex-class I (MHC I), anti-MHC II, antiintercellular adhesion molecule-1 (ICAM-1), and antilymphocyte function-associated antigen-1alpha (LFA-1alpha) antibodies to possibly interfere with DC-thyrocyte interactions. The addition of DC to thyroid follicles clearly inhibited their 3H-TdR uptake, particularly at a 10:1 ratio, in comparison to follicle cultures alone, both under basal conditions and after TSH stimulation (75 +/- 7% and 49 +/- 11% reduction, respectively, n = 4). The follicle T3 secretion (after TSH stimulation) was also suppressed by DC in this system, but to a lesser extent (at best at an 1:1 ratio, 25 +/- 7% reduction, n = 4). The DC-induced inhibition of thyroid follicle growth was totally abrogated after addition of anti-IL-1beta antibodies; anti-IL-6 only had effect on the DC inhibition of non-TSH-stimulated thyrocytes, whereas anti-TNF-alpha demonstrated no effect at all. The antibodies to MHC and to adhesion molecules had also no effect on this DC-induced growth inhibition. The effect of the different anti-cytokine and anti-adhesion antibodies on the T3 secretion from thyroid follicles was not investigated. The clear inhibition of thyrocyte growth by splenic DC (classical antigen-presenting cells) again demonstrates the regulatory role of DC in endocrine systems. Proinflammatory cytokines such as IL-1beta and IL-6 are important mediators in this regulation. The here shown dual role of DC represents a link between the immune and endocrine system, which may form the gateway to the understanding of the initiation of thyroid autoimmune reactions and the thyroid autoimmune phenomena seen in iodine deficiency.

TNF-alpha down-regulates type 1 cytokines and prolongs survival of syngeneic islet grafts in nonobese diabetic mice.

Administration of TNF-alpha to autoimmune diabetes-prone nonobese diabetic mice and biobreeding rats inhibits diabetes development; however, the mechanism(s) of diabetes prevention by TNF-alpha has not been established. We used the model of syngeneic islet transplantation into diabetic nonobese diabetic mice to study the effects of TNF-alpha administration on the types of mononuclear cells and cytokines expressed in the islet grafts and on autoimmune diabetes recurrence. Twice daily i.p. injections of TNF-alpha (20 microg/day) from day 1 to day 30 after islet transplantation significantly prolonged islet graft survival; thus, 70% (16 of 23) of mice treated with TNF-alpha were normoglycemic at 30 days after islet transplantation compared with none (0 of 14) of vehicle-treated control mice. Islet grafts and spleens from TNF-alpha-treated mice at 10 days after islet transplantation contained significantly fewer CD4+ and CD8+ T cells, and significantly decreased mRNA levels of type 1 cytokines (IFN-gamma, IL-2, and TNF-beta) than islet grafts and spleens from control mice. Regarding type 2 cytokines, IL-4 mRNA levels were not changed significantly in islet grafts or spleens of TNF-alpha-treated mice, whereas IL-10 mRNA levels were decreased significantly in islet grafts of TNF-alpha-treated mice and not significantly changed in spleens. TGF-beta mRNA levels in islet grafts and spleens were similar in TNF-alpha-treated and control mice. These results suggest that TNF-alpha partially protects beta cells in syngeneic islet grafts from recurrent autoimmune destruction by reducing CD4+ and CD8+ T cells and down-regulating type 1 cytokines, both systemically and locally in the islet graft.

Phenotypic characterization of mononuclear inflammatory cells in salivary glands of bio-breeding rats

The purpose of this study was to assess whether mononuclear cell abnormalities exist in salivary glands from autoimmune Bio-Breeding (BB) rats. Frozen sections of gland tissues were prepared from five diabetes-resistant BB rats (BB-DR), from five BB rats with diabetes (BB-DP) and from five Wistar rats. A panel of six monoclonal antibodies was used to identify membrane antigens associated primarily with monocytes (ED1), mature tissue macrophages (ED2), lymphoid macrophages (ED3), MHC class II (Ia) antigen (0X6), CD5+ T lymphocytes (0X19), and rat B lymphocytes (0X33). Normal submandibular, sublingual and parotid glands contained few EDI-positive cells, usually two or fewer per field. Tissue macrophages identified by clone ED2 comprised a major mononuclear cell subset in both Wistar and BB rats. However, the number of ED2-positive mononuclear cells was significantly depressed in the submandibular and parotid glands from BB-DR and BB-DP animals, being present in quantities 25–50% of those observed in glands from normal Wistar rats ( p < 0.001). In contrast, 25- to 30-fold greater numbers of ED3-positive macrophages were observed in submandibular glands from BB rats ( p < 0.001). MHC class II (Ia) antigen expression also was 4− to 6-fold greater in BB rat submandibular glands, compared to Wistar rats ( p < 0.001). CD5+ T-lymphocytes were rare or entirely absent in BB sublingual glands (0 to 1 cell per 0.87 mm 2 field), compared to 47 cells per field from Wistar sublingual glands. No B lymphocytes were identified with antibody OX33 in any of the rat strains. These findings indicate that BB rat salivary glands differ significantly from Wistar salivary glands. In BB rats there is a rich population of ED3-positive macrophages and T lymphocytes in submandibular gland, low quantities of T lymphocytes in sublingual gland, and fewer ED2-positive macrophages in all three major salivary glands. These differences in mononuclear cell subpopulations may also influence salivary gland function in mucosal immunity.

Role of macrophages and macrophage-derived cytokines in the pathogenesis of Kilham rat virus-induced autoimmune diabetes in diabetes-resistant BioBreeding rats.

The diabetes-resistant BioBreeding (DR-BB) rat, derived from diabetes-prone forebears, does not normally develop spontaneous insulitis or diabetes, but when infected with Kilham rat virus (KRV) this animal develops autoimmune diabetes similar to the diabetes-prone BioBreeding (DP-BB) rat. In this study, we attempted to determine whether macrophages and macrophage-derived cytokines play a role in the development of KRV-induced diabetes in DR-BB rats. Seventy-eight percent of DR-BB rats treated with KRV and poly(I:C) develop diabetes, whereas depletion of macrophages with liposome-encapsulated dichloromethylene diphosphonate (lip-Cl2MDP) in KRV and poly(I:C)-treated DR-BB rats results in the near-complete prevention of insulitis and diabetes. Measurement of the macrophage-derived cytokines IL-12, TNF-alpha, and IL-1beta revealed a selective increase of their expression, after KRV infection, in the splenic lymphocytes and the pancreatic islets. Measurement of CD4+ T cell-derived cytokines revealed that IL-2 and IFN-gamma cytokine gene expression closely correlates with an elevation of IL-12, but IL-4 and IL-10 do not change. Depletion of macrophages before the isolation of splenic lymphocytes from DR-BB rats treated with KRV and poly(I:C) resulted in the loss of ability to transfer diabetes to young DP-BB rats. On the basis of these observations, we conclude that macrophages and macrophage-derived cytokines play a critical role in the cascade of events leading to the destruction of pancreatic beta cells, culminating in the development of insulin-dependent diabetes mellitus.

The lymphopenia (lyp) gene controls the intrathymic cytokine ratio in congenic BioBreeding rats.

The lymphopenia gene (lyp) on rat chromosome 4 is closely linked to autoimmune diabetes in the BioBreeding (BB) rat. Lyp controls the number of peripheral lymphocytes by reducing T cells of the RT6+ phenotype by almost 90%. Following nine cycle of marker-assisted cross-intercross breeding we have developed congenic lyp/lyp, lyp/+ and +/+ (wildtype) rats on the background of DR rats. Prediabetic and insulitis free lyp/lyp, lyp/+ and +/+ rats were used to determine the effect of lyp on cytokine expression in the thymus. In situ hybridization of thymus cryosections showed that the interferon gamma (IFN gamma) mRNA expression was highest in lyp/lyp rats and the hybridization signal was restricted to the medullary compartment. The frequency of IFN gamma and interleukin (IL)-10 mRNA expressing cells in isolated thymocytes determined by quantitative image analysis, demonstrated an increased IFN gamma: IL-10 ratio in thymocytes from lyp/lyp homozygotes compared to lyp/+ and +/+ rats. This confirmed a lyp gene dose-dependent segregation of the IFN gamma high phenotype. Recombinant human glutamic acid decarboxylase (GAD65) increased the number of IFN gamma and IL-10 mRNA expressing thymocytes after in vitro culture. We conclude that the quantitative ratio of cytokine producing thymocytes is associated with the lyp genotype. These potentially autoreactive thymocytes may explain the establishment of beta-cell directed autoimmunity in the BB rat despite peripheral lymphopenia.

A pronounced thymic B cell deficiency in the spontaneously diabetic BB rat.

In an attempt to elucidate the origin of the T cell lymphopenia and/or the beta-cell-specific autoimmunity observed in diabetes-prone Bio-Breeding (DP-BB) rats, a thymic cDNA library was subjected to differential screening with thymic cDNA probes of DP-BB rats and nonlymphopenic nondiabetic controls. This approach resulted in the identification of a prominent lack of thymic B cells in DP-BB rats. This deficiency is distinct from a less pronounced peripheral B cell deficiency of different timing. The thymic B cell defect is linked to the lymphopenia trait on chromosome 4 and thereby with susceptibility to diabetes in crosses involving the DP-BB rat. In conclusion, our data suggest that the contribution of thymic B cells to the (negative) selection of thymocytes is inadequate in DP-BB rats, thus providing a plausible explanation for at least some of the spontaneous autoimmune phenomena in this animal model.

Induction of insulin-dependent diabetes mellitus in PVG.RT1 u rats

THE BIOBREEDING (BB) rat strain is used to model human autoimmune insulin-dependent diabetes mellitus (IDDM). 1 The diabetes-resistant (DR) subline of the BB rat does not develop diabetes spontaneously, but is genetically predisposed to the disorder. 1 More than 90% of BB-DR rats become diabetic after combined treatment with a depleting monoclonal antibody (MAb) to remove RT6.1 + regulatory T cells and polyinosinic:polycytidylic acid (poly I:C), an inducer of interferons. 2 Lymph node T cells from diabetic BB-DR rats adoptively transfer IDDM to untreated histocompatible (RTI u) WAG- nu/nu rats. 3,4 Thymocytes from either normal or diabetic BB-DR rats also transfer IDDM provided that recipients are treated with anti-RT6.1 MAb to preclude maturation of RT6 + regulatory T cells from the transfused thymocytes. 5 We have previously observed that other rat strains that express the RT6.1 alloantigen, among them the YO (RTI u) and LEW (RT1 1) strains do not develop autoimmune diabetes when treated in the same way as BB-DR rats. 6 To investigate further if combination treatment with anti-RT6.1 MAb and poly I:C can induce autoimmunity in strains other than the BB, we studied major histocompatibility complex (MHC) congenic PVG.RTI u rats. These animals are phenotypically normal and free of spontaneous disease but reportedly develop IDDM after irradiation and thymectomy. 7

Molecular cloning and characterization of the T-cell mono(ADP-ribosyl)transferase RT6. Relationships to other mADPRTs and possible functions.

The RT6 system (1) was first detected and serologically defined on peripheral T cells of different inbred strains of the Norway rat (2-6). It gained particular interest when it was reported that the diabetes-prone BioBreeding rat (DP-BB), which develops insulindependent diabetes mellitus (IDDM) between postnatal day 60 and 120 in a high frequency, does not appropriately express RT6 on the surfaces of its T cells (7). Subsequently RT6 -homologous genes were also detected in mice, primates and some other species (8, 9).

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.

Histomorphological aspects of the development of thyroid autoimmune diseases: consequences for our understanding of endocrine ophthalmopathy.

In this short review we will first evaluate the histomorphological aspects of the human and spontaneous animal thyroid autoimmune diseases. These diseases include Hashimoto goiter, primary myxedema, Graves' disease, and the spontaneous forms of thyroiditis in the Bio Breeding (BB) rat, the nonobese diabetic (NOD) mouse, and the obese strain (OS) of chicken. Based on sequential histomorphological events in the animal models of thyroid autoimmune disease, a mechanism for the pathogenesis of thyroid autoimmune disease is proposed. Since one of the human thyroid autoimmune diseases, specifically Graves' disease, is often associated with ophthalmopathy, the histomorphological aspects of the ophthalmopathic process are also evaluated to consider its possible autoimmune character.

Rat RT6.2 and mouse Rt6 locus 1 are NAD+: arginine ADP ribosyltransferases with auto-ADP ribosylation activity.

RT6 is a glycosylphosphatidylinositol-linked protein found on the surface of mature rat T lymphocytes. Cells that express RT6 have an immunoregulatory function and modulate the expression of autoimmune diabetes mellitus in the BioBreeding rat. A homologue of the rat RT6 gene, designated Rt6, has been identified in the mouse, but expression of mouse Rt6 protein has not been documented. Rat RT6 is known to be a nicotinamide adenine dinucleotide (NAD+) glycohydrolase. We now report that rat RT6.2 and recombinant mouse Rt6 locus 1 proteins possess auto-ADP ribosylation activity. In addition, mouse Rt6 but not rat RT6, catalyzes the ADP ribosylation of exogenous acceptors such as histones. The ADP-ribosyl-protein bonds in auto-ADP-ribosylated rat RT6.2, auto-ADP-ribosylated mouse Rt6, and ADP-ribosylhistone synthesized by Rt6 were stable to HgCl2 and HCl, but labile to NH2OH, consistent with ADP ribosylarginine linkages. To determine if these enzymatic activities could affect the function of rat T cells, the effect of substrate availability on lymphocyte proliferation was examined. An inverse correlation was observed between NAD+ concentration in the medium and the ability of rat T cells to respond to anti-CD3, ConA, and PMA plus ionomycin. The data suggest that lymphocyte surface ADP ribosyltransferases could be involved in signaling and immunoregulatory processes.

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.

Abnormal thymocyte subset distribution and differential reduction of CD4+ and CD8+ T cell subsets during peripheral maturation in diabetes-prone BioBreeding rats.

Abstract In this study we quantified CD8+ and CD4+ T cells in T lymphocytopenic BB rats as compared with control rats at given stages along the maturational pathway from immature thymocytes to mature peripheral T cells. Our results show that BB rats exhibit abnormal thymocyte subset distribution. Numbers of mature TCRhigh/CD4-8+ thymocytes, and also their TCRhigh/CD4+8+ precursors were decreased, as were levels of CD8 expression on all thymocyte subsets investigated. By analogy with mouse thymocyte development, these findings suggest a decreased efficiency for positive selection of CD8 precursors in BB rats. Furthermore, as related to the number of available mature TCRhigh single positive thymocytes, numbers of CD4+ and CD8+ T cells most recently migrated from the thymus were severely decreased in BB blood, indicating either reduced thymic output or rapid cell death after migration. Subsequently, in peripheral blood and cervical lymph nodes, a 95% decrease of CD8+ and a 50 to 80% decrease of CD4+ T cells were demonstrated upon maturation from recent thymic migrants to mature peripheral T cells, leaving the BB rat with a severely reduced T cell population, consisting of CD4+ T cells and a minute population of CD8+ T cells. The vast majority of the latter was found to have an immature peripheral phenotype. Possible consequences of our findings for the generation of autoreactive CD8+ T cells are discussed.

Resistance to glomerular injury in the diabetic biobreeding rat.

This study was designed to determine whether the diabetic BioBreeding rat develops significant renal injury following long-term moderate to severe hyperglycaemia. Diabetic and control rats were followed from the onset of diabetes (2-4 months) to 18 months of age. Frank proteinuria and/or albuminuria were always absent. Glomerular filtration rate, measured by inulin clearance (ml min-1 (100 g body weight)-1), was significantly higher in diabetic rats than in controls at 10, 12 and 18 months of age. Advanced glycosylation end-product cross-links assessed by percentage solubility of tail tendon collagen were moderately increased in diabetic compared with control animals. Urinary excretion of advanced glycosylation end-products in unfractionated urine and in urine fractionated for low molecular mass peptides (< 10 kDa) was 11-fold greater in the diabetic rats than in the control group. Urinary excretion of nitric oxide metabolites (nmol NO2- and NO3- (24 h)-1) were significantly (P < 0.05) greater in diabetic rats than in controls after 8 months of age. Mild histopathology resembling human diabetic nephropathy, including increased mesangial volume and glomerular basement membrane thickness, was detected at 18 months of age. The findings of hyperfiltration and mild glomerular morphological changes in diabetic BioBreeding rats are similar to the abnormalities seen in stage 2 human diabetic nephropathy. We hypothesize that two factors which may contribute to the resistance or tolerance to renal injury in the BioBreeding diabetic rat are increased nitric oxide production and the decreased accumulation of advanced glycosylation end-products.

T cells from BB-DP rats show a unique cytokine mRNA profile associated with the IDDM1 susceptibility gene, Lyp.

Diabetes prone biobreeding rats display several abnormalities in T cell numbers, T cell function and T cell surface phenotype which are associated with the onset of spontaneous disease. One of the most pronounced abnormalities in these animals is a marked T cell lymphopenia which is evident in both CD4+ and CD8+ peripheral T cell subsets. To gain a better understanding as to the nature of T cell responses in these animals, we have utilized RT-PCR to analyze the cytokine mRNA profiles of mitogen activated peripheral T cells derived from lymphopenic and non-lymphopenic animals. Our results suggest that inheritance of the lymphopenia gene, Lyp, is associated with a unique cytokine profile most similar to that previously described for mouse medullary thymocytes. In addition, cell surface staining of peripheral T cells from diabetes prone animals revealed a high frequency of Thyl+ cells, which is characteristic of both thymocytes and recent thymic emigrants. Following thymectomy, T cell responsiveness to a number of different stimuli is greatly reduced on a cell for cell basis as is the absolute number of surviving T cells. Taken collectively, our results suggest that the majority of the peripheral T cell pool in these diabetic prone rats consists of short lived, recent thymic emigrants which most likely also contain the effector cells required for initiation of diabetes.

Renal hypertrophy is associated with upregulation of TGF-beta 1 gene expression in diabetic BB rat and NOD mouse.

Renal hypertrophy is an early feature of diabetes, and it may predispose the kidney to the eventual development of parenchymal dysfunction. Since we have previously demonstrated that short-term culture in high glucose concentration stimulates production of transforming growth factor-beta 1 (TGF-beta 1) in proximal tubular and glomerular mesangial cells, we postulated that this cytokine, which has potent regulatory effects on cellular growth and extracellular matrix production, is important in mediating diabetic renal disease. In this study we evaluated the gene and protein expression of TGF-beta 1 in the kidney of two rodent models of spontaneous insulin-dependent diabetes mellitus [the biobreeding (BB) rat and the nonobese diabetic (NOD) mouse]. In association with the appearance in both models of significant renal hypertrophy, TGF-beta 1 mRNA levels were increased threefold in the kidney of the diabetic BB rat after 3 days of diabetes and also threefold after 7-9 days in the NOD mouse. There was no increase in TGF-beta 1 transcripts in the livers of the diabetic animals, suggesting that this response is tissue specific. Immunohistochemical studies revealed that TGF-beta 1 protein is concordantly elevated in the cortical tubular cells of the diabetic kidney in both models. These results suggest that the stimulated expression of renal TGF-beta is an early manifestation of the involvement of the kidney by diabetes. Whether increased TGF-beta production in the diabetic kidney is a key promoter of diabetic renal manifestations (e.g., hypertrophy) deserves additional studies.

Prevention of autoimmune type I diabetes in biobreeding (BB) rats by a newly established, autoreactive T cell line from acutely diabetic BB rats.

An autoreactive T cell line, BNY-2, was established from lymphocytes isolated from the islets of acutely diabetic BioBreeding (BB) rats after continuous stimulation of the isolated lymphocytes by irradiated lymph node cells. Flow cytometric analysis showed that BNY-2 cells were positive for CD4 and CD5 and were negative for CD8 and RT6, indicating that these cells were phenotypically Th cells rather than cytotoxic T cells. mAbs against rat MHC class II Ags (anti-RT1.D) blocked the proliferation response of BNY-2 cells, suggesting that these cells recognize the MHC class II Du molecule. When splenic T lymphocytes from diabetes-prone BB rats were stimulated by Con A in the presence of irradiated BNY-2 cells or irradiated lymph node cells, the BNY-2 cells had a significant suppressive effect on splenic T cell proliferation, whereas lymph node cells had no effect. When we injected diabetes-prone BB rats i.v. at 30 and 60 days of age with activated BNY-2 cells, the incidence of diabetes was significantly reduced compared with that seen in saline-injected control rats (diabetic incidences were 12 and 80%, respectively). On the basis of these observations, we conclude that autoreactive BNY-2 T cells, established from the pancreatic islets of acutely diabetic BB rats, can prevent the development of autoimmune diabetes in the diabetes-prone BB rat by an immunosuppressive effect.

Evidence that intrathymic islet transplantation does not prevent diabetes or subsequent islet graft destruction in RT6-depleted, diabetes-resistant BioBreeding/Worcester rats.

Pancreatic islet allografts transplanted intrathymically are accepted and restore normoglycemia in streptozotocin-diabetic rats given one injection of antilymphocyte serum. Intrathymic allografts similarly restore normoglycemia in diabetes-prone (DP) Bio-Breeding (BB) rats that have developed spontaneous autoimmune diabetes. Intrathymic islets also reduce the frequency of subsequent diabetes when transplanted prophylactically into young DP rats. These findings suggest that intrathymic transplantation can prevent not only allograft rejection, but also the appearance and recurrence of autoimmune tissue destruction. To explore these hypotheses further, we attempted both to confirm previous studies and to extend them to another model of autoimmune diabetes, the RT6-depleted diabetes-resistant (DR) BB rat. Fewer than 1% of DR-BB rats develop spontaneous diabetes, but most become hyperglycemic after in vivo immune elimination of RT6+ T cells. Using the protocols described in the literature, we observed the following: (1) Consistent with previous reports, intrathymic islet allografts survived indefinitely in streptozotocin-diabetic, antilymphocyte serum-treated, non-BB recipient rats. (2) Consistent with previous reports, intrathymic islet grafts produced long-term normoglycemia in diabetic DP-BB rats and also reduced the frequency of spontaneous diabetes in young animals transplanted prophylactically. (3) In contrast, intrathymic islets (iso- and allografts) neither prevented nor reversed diabetes in RT6-depleted DR rats. We hypothesize that intrathymic islet grafts survive in DP-BB rats because they are lymphopenic and immunocompromised, whereas immunocompetent diabetic DR rats successfully recapitulate the autoimmune disease process. Although intrathymic allograft transplantation is postulated to induce a state of tissue-specific tolerance, our results indicate that this tolerant state may not extend to autoimmune destruction of either isografts or allografts.