Iain L. Campbell

Overexpression of class I major histocompatibility complex accompanies insulitis in the non-obese diabetic mouse and is prevented by anti-interferon-γ antibody

SummaryOverexpression of class I major histocompatibility complex (MHC) proteins on pancreatic islet cells is a characteristic of autoimmune Type 1 (insulin-dependent) diabetes mellitus in humans and in animal models. Studies of post-mortem pancreases from humans with Type 1 diabetes suggest that overexpression of class I MHC proteins may precede mononuclear cell infiltration of the islets (insulitis). Pancreatic histology from the earliest stages of human Type 1 diabetes is rarely available. We have used the non-obese diabetic mouse, given cyclophosphamide to accelerate Betacell destruction, to investigate the temporal relationship between the Overexpression of class I MHC protein and mRNA and other pathological changes associated with Beta-cell destruction. Prior to cyclophosphamide, immunoperoxidase staining showed that expression of class I MHC proteins was greater on islet cells and infiltrating inflammatory cells of the non-obese diabetic mouse than on islet cells of other mouse strains, whereas staining on exocrine cells was similar. On day three after cyclophosphamide administration, when insulitis had regressed, islet class I MHC protein expression had diminished. A dramatic increase in class I MHC protein expression occurred between days seven and nine, concomitant with reinfiltration of the islets by mononuclear cells; Overexpression was seen both on islet cells and on surrounding exocrine cells, but only in the presence of mononuclear cell infiltration. By day 21, class I MHC protein Overexpression was again confined to the islets, the exocrine pancreas being free of infiltration. Class I mRNA also increased dramatically by day eight but had virtually returned to normal by day 12. Overexpression of class I MHC protein following cyclophosphamide was prevented by administration of antiinterferon- γ antibody. Expression of class II MHC proteins was not detected on pancreatic cells following cyclophosphamide but was present on infiltrating mononuclear cells. These findings demonstrate a close association between class I MHC protein and mRNA Overexpression and insulitis in non-obese diabetic mice given cyclophosphamide. They are consistent with the view that class I MHC Overexpression is effected by cytokines secreted by activated immunoinflammatory cells. Class I MHC Overexpression should enhance targeting of cytotoxic T cells to Beta cells bearing autoantigen.

Reduction in insulitis following administration of IFN-γ and TNF-α in the NOD mouse

Abstract In insulin dependent diabetes mellitis (IDDM) beta cell destruction is associated with infiltration of the pancreatic islets by T lymphocytes and macrophages. Cytokine products from the infiltrating immunocytes not only have powerful immunoregulatory actions but also are capable of impairing islet cell functions and have thus been postulated to assume a central role in mediating anti-beta cell immunity and beta cell destruction. In an effort to explore further the role of cytokines in the pathogenesis of IDDM, we examined clinical, metabolic and pathological features of NOD/Wehi mice injected intraperitoneally with multiple doses of IFN-γ and/or TNF-α. Blood glucose profiles were not significantly altered by injection of cytokines alone or in combination. Except for a hypoglycaemic rebound in mice injected with TNF-α, arginine stimulation tests revealed no disturbances in islet secretory function in cytokine injected mice. Compared with vehicle and cytokines alone, injection of IFN-γ + TNF-α was associated with a variety of clinical and pathological changes including abdominal distention, piloerection, ascites, oedema, thymic atrophy, splenic enlargement and pancreatic distention. Histological examination of the pancreas in these mice revealed moderate to severe pancreatitis which included focal haemorrhagic necrosis, oedema and polymorphonuclear and mononuclear cell infiltration. The islets in these mice appeared normal morphologically and when stained for insulin. The injection of IFN-γ + TNF-α, and to a lesser extent TNF-α alone, was associated with a significant reduction in the severity of insulitis. Examination of pancreatic MHC-class I and class II molecule expression revealed in mice given IFN-γ + TNF-α, as compared with controls, significant and uniform induction of both these molecules on ductal and acinar cells; low level MHC-class II expression was also detectable on beta cells in these mice. MHC-class I molecules which were expressed at high levels by beta cells in control mice did not appear to change following administration of the cytokines alone or in combination. We conclude that despite their immunostimulatory actions in vitro and in other models in vivo, systemic administration of the cytokines IFN-γ and/or TNF-α to NOD/Wehi mice does not activate or enhance, and may actually suppress, anti-beta cell immunity in this model.

Characterization of pancreatic T lymphocytes associated with beta cell destruction in the non-obese diabetic (NOD) mouse.

Pancreatic beta cell destruction in the non-obese diabetic (NOD) mouse is mediated by T lymphocytes and macrophages and accelerated by cyclophosphamide. We purified pancreatic T lymphocytes from the NOD mouse for comparative phenotypic and functional analysis with T lymphocytes from spleen, peripheral blood and regional lymph nodes. Pancreatic T lymphocytes from NOD-Wehi mice, which have an incidence of spontaneous diabetes of less than 5%, had a CD4:CD8 ratio of 1.25 +/- 0.23 compared with 2.44 +/- 0.31 for peripheral blood lymphocytes. After cyclophosphamide, the CD4:CD8 ratio of pancreatic lymphocytes increased to 2.30 +/- 0.24 at day 7. T lymphocytes bearing IL-2 receptors increased two- to three-fold in number and their secretion of GM-CSF/IL-3 and IFN-gamma increased to a maximum on day 7. Pancreatic insulin content and mRNA levels declined sharply between days 10 and 12, at which time the majority of pancreatic T lymphocytes in hyperglycaemic mice were CD8+ (CD4:CD8 ratio 0.63 +/- 0.04 compared to 4.14 +/- 1.05 in peripheral blood). The pancreatic T lymphocyte CD4:CD8 ratio in prediabetic NOD-Lt mice, which have an incidence of spontaneous diabetes of about 60% at 150 days, was similar to that in untreated NOD-Wehi mice, but 25% of their pancreatic CD8 T lymphocytes were IL-2-receptor positive. Thus, significant changes in the phenotype of NOD pancreatic T lymphocytes following cyclophosphamide were not reflected in peripheral blood or spleen T lymphocytes. The earliest change after cyclophosphamide was an increase in activated, predominantly CD4+ T lymphocytes; with the development of beta cell destruction and hyperglycaemia, pancreatic T lymphocytes were, as in human IDDM, predominantly CD8+.

Interferon-γ: pleiotropic effects on a rat pancreatic beta cell line

Abstract We have recently shown that interferon-y (IFN-y) markedly upregulates the expression of the class I major histocompatibility proteins on pancreatic beta cells and have therefore postulated that interferon-y may enhance cytotoxic lymphocyte-mediated beta cell damage in insulin-dependent diabetes mellitus. To further explore the interaction between interferon-γ and the pancreatic beta cell we have used the RIN-m5F insulinoma line to define the effects of interferon-γ on major histocompatibility protein expression, (pro)insulin and protein synthesis and cell growth. Interferon-γ induced a dose-dependent increase in the expression of the class I major histocompatibility proteins on the RIN-m5F cells, the maximal increase (10-fold) being seen at an interferon-γ concentration of 1 U/ml. The induction of class I proteins by interferon-γ was nearly completely abolished by cycloheximide. Expression of class II (la) proteins was not detected either in the presence or absence of interferon-y. (Pro)insulin and protein synthesis were decreased by 60% and 40%, respectively, in RIN-m5F cells cultured with interferon-γ (10 U/ml). Furthermore, the growth of RIN-m5F cells was significantly inhibited, and corresponding changes in cell morphology were evident, after 3 days of exposure to interferon-y (10 U/ml). These findings indicate that, in addition to its potential role in amplifying cytotoxic T cell activity against the pancreatic beta cell, IFN-y may also directly inhibit beta cell function and growth. Several mechanisms could therefore account for an ability of IFN-γ to compromise beta cell function and contribute to the pathogenesis of insulin-dependent diabetes.

A new view of the beta cell as an antigen-presenting cell and immunogenic target.

Cellular autoimmunity is thought to be primarily responsible for the selective destruction of islet beta cells in Type I diabetes. Why the T lymphocyte reacts to self and recognizes the beta cell as foreign, as against the other endocrine islet cells, is unknown. One key issue is whether the beta cell itself is capable of presenting autoantigen(s) and thereby breaking T lymphocyte tolerance. In this paper we discuss current concepts of antigen presentation and relate these to recent findings from our laboratory, suggesting that the beta cell can be induced to display many of the phenotypic properties of classical antigen-presenting cells, including induction of MHC and ICAM-1 expression and production of IL-6. Finally, a model is presented which provides a new view of the initiation and perpetuation of autoimmune beta-cell destruction in Type I diabetes.

Inherent beta-cell dysfunction induced by transgenic expression of allogeneic major histocompatibility complex class I antigen in islet cells.

Insulin-dependent diabetes mellitus (IDDM) is generally believed to be an autoimmune disease resulting from T-cell dysfunction that produces beta-cell damage, but it is conceivable that some forms of IDDM are not immunologically mediated. The effect of the expression of a foreign transgenic MHC class I antigen (H-2Kb), restricted to pancreatic islet beta-cells, was tested in vitro and in nude (athymic) mice to determine whether beta-cell dysfunction was due to non-immune mechanisms. The models used clearly excluded immune involvement in beta-cell damage. Fetal pancreas from transgenic and littermate control mice was maintained in organ culture for up to 18 days and insulin secretion into the medium assessed. For the initial 3-4 days in vitro, fetal control and transgenic pancreas secreted similar amounts of insulin, but thereafter insulin secretion by the transgenic tissue decreased in comparison with the controls. When the cultured pancreas was transplanted into nude mice, the transgenic issue produced smaller grafts than the control pancreas, but there was wide variation in graft size. Expression of H-2Kb antigens in beta-cells of nude transgenic mice also resulted in early-onset diabetes. The insulin content in the pancreas of young H-2Kb transgenic euthymic mice, (previously shown not to have insulitis), was reduced but glucagon content was normal. The reduction in in vivo insulin production was similar chronologically to the reduced insulin production by transgenic islets in vitro. These data confirm the non-immune loss of beta-cell function in MHC-transgenic mice and they may be a model for atypical Type I diabetes.

Lack of specificity of islet cell surface antibodies (ICSA) in IDDM

Human islets were isolated by collagenase digestion and tissue culture from pancreata obtained from organ donor subjects and dispersed islet cells were prepared from hand-picked islets. Islet cell surface antibodies (ICSA), detected by indirect immunofluorescence on isolated islet cells, were present in sera from nine of 22 (41%) subjects with recent-onset insulin-dependent diabetes mellitus (IDDM) and three of 11 (27%) control subjects. Sera had been heat inactivated, adsorbed against a human B lymphoblastoid cell line (IM-9) and tested in the presence of 4% bovine serum albumin. However, with a double labelling technique, we were unable to show that ICSA were specific for beta cells. Of the nine ICSA-positive IDDM sera, three stained both beta and non-beta cells, three beta cells only and three non-beta cells only; the three ICSA-positive control sera stained both beta and non-beta cells. There was no apparent relationship between ICSA and standardised measurements of islet cell antibodies (ICA) and insulin autoantibodies (IAA). These results lead us to question whether, despite previous reports, ICSA are specific for beta cells or indeed for IDDM.

Major Histocompatibility Complex Molecules and the Beta Cell: Inferences from Transgenic Models

Type 1 or insulin-dependent diabetes mellitus is a syndrome of chronic hyperglycaemia secondary to insulin deficiency due to the selective destruction of the pancreatic islet β cells. The molecular pathology of the disease in humans is still poorly understood, but a substantial body of evidence favours the view that the β cells are destroyed by an autoimmune process initiated in genetically predisposed individuals by environmental agents such as viruses or chemotoxins (EISENBARTH 1986; ROSSINI et al. 1988; CAMPBELL and HARRISON 1989). Evidence in humans for the autoimmune basis of type 1 diabetes includes the following: (a) the presence of circulating autoantibodies to islet cells and/or insulin in the majority of newly diagnosed patients (GLEICHMANN and BOTTAZZO 1987), (b) mononuclear cell infiltration or “insulitis” of islets seen in the pancreas obtained at autopsy from newly diagnosed subjects (GEPTS and LE COMPTE 1981; FOULIS et al. 1986), (c) recurrence of diabetes associated with insulitis in recipients of pancreas isografts from major histocompatibility complex (MHC) identical siblings (SIBLEY et al. 1985) and (d) an increase in the frequency and duration of remissions from insulin dependence in newly diagnosed patients treated with immunosuppressive agents (HARRISON et al. 1985; ASSAN et al. 1985).

A2B5-reactive ganglioside expression determines the differentiation stage and capacity of rat insulinoma (RIN) sublines

We have generated rat insulinoma (RIN) sublines AlGh, m5F, A12, A13 and AhGh with increasing surface expression of the A2B5 ganglioside, a marker of differentiation. We asked whether the capacity of the sublines to differentiate was related to their stage of differentiation, as is characteristic of cells within the normal beta-cell lineage. To answer this, we measured the effect of the differentiation inducer sodium butyrate (NaB, 1 mM) on proliferation, insulin content, secretion and biosynthesis, and the expression of A2B5 and 3G5 gangliosides by the sublines. Six days after exposure to NaB, cell numbers/dish ranged from (1-3) x 10(6) compared to (4-6) x 10(6) in control cultures. By day 2, AlGh, m5F, A12, A13 and AhGh cells exposed to NaB contained 1.5-, 1.4-, 1.4-, 1.2- and 1.0-fold higher amounts of insulin, respectively, and by day 6, 3.6-, 2.3- and 1.0-fold higher, and 1.2- and 2.4-fold lower, amounts of insulin, respectively, than control cells. After 2 days, insulin secretion from AlGh, m5F, A12, A13 and AhGh cells was 1.7-, 1.0-, 1.5-, 1.0- and 1.0-fold higher, respectively, and the rate of (pro)insulin biosynthesis 1.7-, 2.3-, 1.3-, 1.0- and 1.0-fold higher, respectively, than control cells. After 6 days, A2B5 ganglioside expression was increased 3-, 1.9- and 2-fold on m5F, A12 and A13 cells, respectively, but was not significantly altered on AlGh and AhGh cells. 3G5 ganglioside expression was increased 1.5- and 8.4-fold, respectively, on AlGh and m5F cells, but was unaltered on A12, A13 and AhGh cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Murine models of autoimmune diabetes: nonspecific cytotoxic lymphocytes derived from pancreatic islets in the presence of IL-2.

Our aim was to derive T lymphocyte lines that specifically recognize islet antigens in murine models of autoimmune diabetes. Islets of Langerhans infiltrated with lymphocytes were isolated either from mice previously injected with multiple low doses of streptozotocin or from NOD-WEHI mice and were cultured in the presence of the T cell growth factor, interleukin 2 (IL-2). With islets from both models of autoimmune diabetes, rapidly proliferating, large granular lymphocytes emerged after 7-10 days and destroyed the islets and other cells such as fibroblasts in the cultures. Cytotoxicity assays showed that these cells were capable of destroying both P815 and YAC-1 tumor cells. In contrast to lymphocytes present initially in the islet infiltrates which express predominantly the L3T4 marker, the large granular lymphocytes were shown to be Ly-2 positive. They also expressed the alpha beta T cell receptor and contained mRNA for the alpha beta T cell receptor demonstrable by in situ hybridization. While morphologically similar to NK cells these large granular lymphocytes bear T cell markers and destroy a broader range of targets. They may represent a minor population of T lymphocytes particularly responsive to IL-2 although other studies show that T cells generally can develop a similar phenotype after prolonged culture with IL-2. The lack of target cell specificity indicates that these IL-2-stimulated large granular lymphocytes are unlikely to mediate the immunopathogenesis of diabetes in these animal models.