DR-BB Rats Research Articles

High Frequency Apoptosis of Recent Thymic Emigrants in the Liver of Lymphopenic Diabetes-Prone BioBreeding Rats

Diabetes-prone (DP) BioBreeding (BB) rats develop spontaneous autoimmune diabetes. DP-BB thymocyte export is reduced, and most thymic emigrants disappear rapidly from peripheral lymphoid tissues. DP-BB rats are consequently lymphopenic and circulate severely reduced numbers of T cells. Peripheral T cells present are phenotypically immature (Thy1+) and appear activated. We hypothesized that DP-BB recent thymic emigrants have a shortened life span and disappear by apoptosis. The percentage of T cells with an alphabetaTCR(low) B220+ CD4- CD8- phenotype was increased in DP peripheral lymphoid tissues when compared with normal, nonlymphopenic diabetes-resistant (DR) BB rat tissues. There was no evidence of DNA fragmentation in freshly isolated DP- or DR-BB rat cells, but, after 24 h of culture, a higher proportion of DP- than DR-BB splenic T cells underwent apoptosis. We then tested the hypothesis that BB rat T cells with the alphabetaTCR(low) B220+ CD4- CD8- phenotype accumulate and undergo apoptosis in the liver. Such cells were observed undergoing apoptosis in both DP- and DR-BB rats, but comprised approximately 80% of intrahepatic T cells in DP vs approximately 20% in DR-BB rats. Most alphabetaTCR(low) B220+ CD4- CD8- cells in the liver were also Thy1+. The data suggest that T cell apoptosis in the DP-BB rat is underway in peripheral lymphoid tissues and is completed in the liver. Increased intrahepatic apoptosis of recent thymic emigrants appears in part responsible for lymphopenia in DP-BB rats and the concomitant predisposition of these animals to autoimmunity.

Paradoxical Antidiabetogenic Effect of γ-Interferon in DP-BB Rats

Previous studies have shown that anti-gamma-interferon (IFN-gamma) antibody reduces the frequency of autoimmune IDDM in the DP-BB rat. We tested the effects of systemically administered recombinant rat IFN-gamma in both DP-BB and DR-BB rats. Unexpectedly, IFN-gamma markedly reduced the incidence of IDDM as compared with control rats when administered six times per week at a dosage of 280,000 U between ages 30-35 to 105 days or ages 60-64 to 105 days. A lower dosage (28,000 U on alternate days) was also protective when administered to DP-BB rats between birth and age 60 days. However, long-lasting protection against IDDM development over the 1-year study period was achieved only by the highest dosage of IFN-gamma administered from age 30 to 105 days. Ex vivo production of tumor necrosis factor-alpha from splenic lymphoid cells (SLCs) and peritoneal macrophages of the rats treated with IFN-gamma was comparable with that of controls; however, SLCs from the IFN-gamma-treated animals secreted lower amounts of IFN-gamma after stimulation with concanavalin A. IFN-gamma treatment also markedly reduced the frequency of phenotypically activated SLC-expressing class II antigens and interleukin-2 receptor. Finally, in agreement with the observed antidiabetogenic effects, exogenously administered IFN-gamma induced neither insulitis nor IDDM development in DR-BB rats, a subline of DP-BB rats in which autoimmune diabetes rarely occurs spontaneously but can be induced by administration of polyinosinic-polycytidilic acid.

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

Anti-endothelial cell autoantibodies in BB rats with spontaneous and induced IDDM.

Anti-endothelial cell (anti-EC) antibodies occur in several autoimmune diseases, including human IDDM, but the time course of their development and their importance in disease pathogenesis are unknown. To study such antibodies further, we investigated the BB rat model of autoimmunity. Diabetes-prone (DP) BB rats spontaneously develop autoimmune diabetes, whereas coisogenic diabetes-resistant (DR) BB rats are disease free but can be induced to become diabetic by the depletion of T-cells expressing the RT6 alloantigen. Anti-EC autoantibodies were readily detectable in both untreated DP-BB rats and RT6-depleted DR-BB rats before the onset of diabetes. Their concentration increased with time. The anti-EC antibodies in DP-BB rats were almost exclusively of the IgG2b subclass, whereas those in RT6-depleted DR-BB rats included both the IgG1/2a and the IgG2b subclasses. We also found that intravenous injections of purified immunoglobulins from RT6-depleted DR-BB rats induced abnormal pancreatic vascular leakage in mice. The preabsorption of immunoglobulins against cultured ECs abolished this activity. The pretreatment of mice with silica also abolished the ability of immunoglobulins of RT6-depleted DR-BB rats to induce pancreatic leakage, suggesting that monocytes are involved in the mechanism of anti-EC autoantibody-induced vascular leakage. We conclude that anti-EC autoantibodies are present in rat strains that are genetically predisposed to develop autoimmune diabetes. Their presence early in the disease process and their ability to induce pancreatic vascular leakage suggest that they may participate in diabetes pathogenesis.

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.

DR-BB Rat Thymus Contains Thymocyte Populations Predisposed to Autoreactivity

We have induced autoimmune insulin-dependent diabetes mellitus (IDDM) in athymic WAG rats by transfusing thymocytes from histocompatible phenotypically normal rats of the DR-BB strain. DR-BB rats rarely develop spontaneous IDDM, but readily become hyperglycemic if depleted in vivo of regulatory T-cells that express the RT6.1 maturational alloantigen. Successful adoptive transfer of IDDM by DR-BB thymocytes required that the athymic recipients be depleted of emerging populations of donor-origin RT6.1+ T-cells. Thymocytes from both normal and RT6-depleted diabetic DR donors were equally capable of transferring autoimmunity. In contrast, thymocytes from normal histocompatible YOS rats failed to transfer IDDM. The autoreactive potential of DR-BB rat thymocytes was minimal from birth to 4 weeks of age and then increased substantially at 8-9 weeks of age. These results demonstrate that the DR-BB rat thymus harbors abnormal cell populations predisposed to autoreactivity. The data localize the developmental defect leading to diabetes in the BB rat to an abnormal intrathymic selection process.

DR-BB rat thymus contains thymocyte populations predisposed to autoreactivity

DR-BB rat thymus contains thymocyte populations predisposed to autoreactivity

Adoptive Transfer of Autoimmune Diabetes Mellitus to Athymic Rats: Synergy of CD4+ and CD8+ T Cells and Prevention by RT6+ T Cells

We describe the induction and prevention of autoimmune insulin dependent diabetes mellitus (IDDM), and its pathological substrate, insulitis, in congenitally athymic nude rats following injections of major histocompatibility complex (MHC) compatible lymph node T cells. The cells capable of adoptive transfer of autoimmunity were obtained from diabetes resistant (DR) BB rats that had been rendered hyperglycemic by in vivo depletion of the RT6+ regulatory T cell subset. We first established that our adoptive transfer assay system is cell dose- and time dependent and therefore amenable to quantitative analysis. It was also observed that both CD4+ and CD8+ T cells are required for efficient transfer of autoimmunity. The data indicate that, as in the NOD mouse, a synergistic interaction between CD4+ and CD8+ T cells is important for beta cell destruction. Finally, we demonstrated that the admixture of equal numbers of lymph node T cells, 60% of which were RT6+, from intact, non-diabetic DR rats prevented the adoptive transfer of IDDM mediated by diabetogenic T cells from RT6-depleted DR-BB rats. We conclude that an equilibrium between autoreactive and regulatory cells determines the expression of autoimmunity in the DR-BB rat and in the adoptive transfer of diabetes in quantitative analytical systems.

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.

Diabetes manifestation in BB rats is preceded by pan-pancreatic presence of activated inflammatory macrophages.

Pancreata of normoglycemic diabetes-prone (dp) and diabetes-resistant (dr) BB rats and normal Wistar rats were screened for macrophage infiltration by immunohistochemistry and by electron microscopy. Inflammatory macrophages were found in the endocrine as well as the exocrine part of the pancreata from dp BB rats. In the exocrine tissue they had a different phenotype (ED1+, ED2+, W3/25+, Ox17+) from that found in pancreata from dr BB and Wistar rats (predominantly ED1-, ED2+, W3/25+, Ox17-). The number of macrophages in exocrine portions of pancreata from the various rat strains were not different. By electron microscopy scattered macrophage-associated tissue lesions and phagocytosis of cell debris were found throughout the exocrine tissue and in islets of dp BB rats. Such lesions were low or absent in biopsies of animals that later did not develop diabetes. We conclude that macrophage-mediated cytotoxicity during the early phases of diabetes development in BB rats is not restricted to islets, but is a generalized, pan-pancreatic event.

Altered Expression of Diabetes in BB/Wor Rats by Exposure to Viral Pathogens

Autoimmune diabetes mellitus affects greater than 50% of diabetes-prone BB (DP BB) rats but less than 1% of diabetes-resistant BB (DR BB) rats. We report an outbreak of spontaneous diabetes among DR BB rats that coincided with serologic evidence of the onset of viral infection. This apparent link between a change in the environment and the expression of diabetes then led us to study the interaction of environmental exposure to viral pathogens in this disorder with virally seropositive and seronegative populations of BB rats and polyinosinic-polycytidylic acid (poly I:C), an interferon inducer known to accelerate diabetes onset in DP rats. We administered a cytotoxic anti-RT6 monoclonal antibody, poly I:C, or both to DR rats. Depletion of the RT6.1+T-lymphocyte population has previously been shown to induce diabetes and thyroiditis in DR rats. RT6 alone did not induce diabetes in seronegative DR rats, and poly I:C was only weakly effective, but nearly all animals given both reagents became diabetic. When given to seropositive DR rats, either reagent alone induced diabetes; when given to non-BB rats, neither agent was effective. Poly I:C also accelerated the onset of DP diabetes to a greater extent in seropositive than in seronegative rats. We conclude that expression of the genetic predisposition to diabetes present in all BB rats depends on cellular factors that include the presence or absence of regulatory (RT6+) T lymphocytes and modulatory environmental factors including exposure to viral pathogens.

Induction of Diabetes by Polyl:C and Anti-RT6.1 Antibody Treatment in DR-BB Rats.

To determine whether environmental factors could affect the incidence of diabetes in RT6.1+ lymphocytes-depleted diabetes resistant (DR) BB rats, we tested polyinosinic-polycytidylic acid (Poly I:C), as an immune activator, in conjunction with anti-RT6.1 antibody in DR-BB rats which were bred in a specific pathogen free (SPF) condition. Diabetes was induced by the combined administration of poly I:C and anti-RT6.1 antibody. The use of poly I:C or anti-RT6.1 antibody alone did not cause diabetes. These results suggest that RT6.1+ T lymphocytes regulate autoimmune diabetes and that non-specific immune activation caused by environmental factors plays a key role in inducing diabetes in DR-BB rats.

Cytotoxic T lymphocytes do not control lymphocytic choriomeningitis virus infection of BB diabetes-prone rats

BioBreeding Worcester diabetes-prone (BBdp) rats develop insulin-dependent autoimmune-driven diabetes mellitus spontaneously and intravenous administration of 1 x 10(7) p.f.u. of lymphocytic choriomeningitis virus (LCMV) to young adult mice prevents disease. The virus is lymphotropic, binding to and replicating in such cells. BBdp rats fail to generate virus-specific major histocompatibility complex-restricted cytotoxic T lymphocyte (CTL) responses when challenged with this dose or other doses of LCMV, Pichinde virus or vaccinia virus. Yet such rats clear virus effectively and show no evidence of persistent infection. Associated with this clearance of virus and establishment of immunity is the production of neutralizing antibodies. In contrast, diabetes-resistant (BBdr) rats generate virus-specific CTL responses. Furthermore LCMV binds to fewer lymphoid cells of BBdr rats (in comparison to those of BBdp rats) and replicates in fewer lymphocytes (by one order of magnitude) from these rats. Thus, unlike mice in which CTLs play a dominant role in the control of LCMV infection, BBdp rats do not overcome this infection via CTLs. In addition, both the BBdp rats and their BBdr counterpart may provide useful models for determining whether or how individual lymphocyte subsets function in the induction of CTL responses, for the analysis of virus-induced immunosuppression and for the use of viruses or their products as therapeutic modalities.

The pathogenesis of autoimmune diabetes mellitus

There is increasing evidence that both DP and DR BB rats fail to clonally delete autoreactive T cells in the thymus that are important in the development of autoimmune IDDM. The DP BB rat also has a defect in its ability to generate a regulatory (RT6+) T-cell population that would prevent the onset of diabetes and, therefore, it becomes spontaneously diabetic. The DR rat develops autoreactive T cells, but does not express diabetes because of the concurrent development of a regulatory (RT6+) T-cell population. We suggest that in the BB rat, the initial immunological lesion is orchestrated by an APC in close proximity to pancreatic islet beta cells, and may be specifically directed to the beta cell itself. The release of cytokines in the vicinity of the beta cell destroys this highly susceptible target, causing the release of beta cell 'autoantigens'. These autoantigens, in turn, target autoreactive T cells to the beta cells, allowing a focal destructive process to spread throughout the pancreas. The ultimate destruction of the islets and the development of diabetes result from a cascading effect of this process, with the recruitment of other non-specific immune mediators. A similar process may also be initiated by APC within the thyroid of the rat, resulting in thyroiditis. The fact that the thyrocyte does not die is unexplained, but it could relate to the relative insensitivity of this cell type to various cytokines.

Prevention of diabetes in the BB rat by essential fatty acid deficiency. Relationship between physiological and biochemical changes.

Essential fatty acid (EFA) deficiency exerts a striking protective effect in several animal models of autoimmune disease. We now report that EFA deprivation prevents diabetes in the BB rat, an animal model of human insulin-dependent diabetes mellitus. In diabetes-prone (DP)-BB rats, the incidences of spontaneous diabetes and insulitis (the pathological substrate of autoimmune diabetes) were greatly reduced by EFA deficiency. This beneficial effect of the deficiency state was also seen in diabetes-resistant (DR)-BB rats that, after treatment with antibody to eliminate RT6+ T cells, would otherwise have become diabetic. The susceptibility of EFA-deprived DP-BB rats to spontaneous diabetes was restored when they were given dietary supplements of linoleate at 70 d of age (during the usual period of susceptibility), but not when they were repleted beginning at 120 d (after the peak incidence of diabetes). EFA deficiency did lead to growth retardation, but calorically restricted control rats demonstrated that the protective effect of the deficiency state was not a function of decreased weight. To examine the relationship between the biochemical changes of EFA deficiency and its physiological effects in this system, we compared the fatty acid changes that occurred in EFA-deficient animals that did and did not develop diabetes. Nondiabetic animals had significantly lower levels of (n-6) fatty acids (i.e., linoleate and arachidonate) and higher levels of oleate, an (n-9) fatty acid, than did diabetic animals. Levels of 20:3(n-9), the fatty acid that uniquely characterizes EFA deficiency, were similar in both groups, however. Among diabetic EFA-deficient rats, the age at onset of diabetes was found to correlate inversely with the level of (n-6) fatty acids, the least depleted animals becoming diabetic earliest, whereas there was no correlation with levels of 20:3(n-9). Among animals repleted with linoleate beginning at 70 d, restoration of susceptibility to diabetes correlated with normalization of the level of arachidonate. In summary, EFA deprivation reduced the frequency of diabetes in both DP and RT6-depleted DR-BB rats. This protective effect was strongly associated with depletion of (n-6) fatty acids, particularly arachidonate, but not with accumulation of the abnormal 20:3(n-9). Conjecturally, arachidonate and/or a metabolite may play a key role in mediating inflammatory injury in this animal model of autoimmune diabetes.

Unaltered Class II Histocompatibility Antigens and Pathogenesis of IDDM in BB Rats

The BB rat spontaneously develops insulin-dependent diabetes mellitus (IDDM) as an autoimmune abnormality involving the class II molecules of the major histocompatibility complex (MHC). The rat MHC (RT1 complex) encodes two class II loci, RT1.B and RT1.D. The possibility that variant or unique class II MHC molecules may be associated with IDDM susceptibility was directly examined by determining the nucleotide sequences of class II mRNAs and/or cDNAs from the diabetes-prone (DP) BB rat, the diabetes-resistant (DR) BB rat, the normal histocompatible Wistar-Furth (WF) rat, and the Lewis rat. Sequence analysis indicates that the beta-chains of the RT1.B and RT1.D molecules of the u haplotype from DP-BB, DR-BB, and WF rats are identical but that they are different from other rat alleles and published mouse class II sequences. At the nucleotide level, the NH2-terminal domain of RT1.D beta of the BB and WF rats differs by a single silent nucleotide substitution. Comparisons with the sequences of the Lewis rat indicate hypervariable allelic differences and that the u and I haplotypes are remarkably similar. These findings establish that the class II molecules of the DP-BB rat are not variant or unique and that unaltered class II molecules of the u haplotype support the autoimmune response in the BB rat.