Type 1 Diabetes: Cellular, Molecular & Clinical Immunology
Theoretical Essay A - The Development of Autoimmune Diabetes: Theoretical Aspects
Kevin J. Lafferty
The nonobese diabetic (NOD) mouse develops type I diabetes spontaneously and the disease is associated with lymphocytic infiltration of the pancreatic islet and eventual destruction of islet b-cells. This destructive process is quite specific, with a-cells and other cellular components of the pancreatic islets initially being spared. The disease process represents a form of tissue-specific autoimmunity. It is associated with the development of antibodies specific for islet components such as insulin glutamic acid decarboxylase (GAD) and other islet-cell antigens (see Chapter 2). Although such antibodies become elevated with the development of disease, there is no evidence that antibodies of themselves are pathogenic.1 The disease process appears to represent a form of cell-mediated immunity dependent on CD4 T cell activity, and it has been demonstrated that disease develops in mice whose capacity to produce humoral immunity has been blocked.1
Initiation of Disease
The specificity of the disease process resulting in damage to only the b-cells of the islet suggests that the pathogenic process may involve a cytotoxic T cell attack directed at some b-cell-specific antigens. The development of disease is dependent on the activity of both CD4 and CD8 T cells2,3 and it is reasonable to suggest that the CD4 cell is behaving as a helper cell required for the activation of the cytotoxic CD8 T cell that in turn initiates islet damage. However, studies carried out at the Barbara Davis Center demonstrated that although both CD4 and CD8 T cells are required for the initiation of disease, the immunological specific effector cell is the CD4 T cell (see Chapter 5).3,4 Pancreatic islet-reactive T cell clones have been isolated from the NOD mouse and in all cases these cells have proved to express the CD4 phenotype (see Chapter 4). Such cloned CD4 T cells have the capacity to initiate disease in the absence of CD8 T cell function.5,6 Thus, while there is no question that the CD8 T cell plays an essential role in the development of disease, this cell is not appear to be immunologically specific effector cell. Ann Cooke has shown this cell to be required early in the process of disease development, indicating that the CD8 T cell has an accessory or helper function in the activation of the CD4 effector of the autoimmune response.7 There are examples were CD8 T cells can modify the activation of CD4 T cells and this process is mediated via active involvement of the antigens-presenting cell (APC).8 There is also evidence9 that the early activated CD8 T cell produces high levels of interferon gamma (IFN-g) he and in this way forces the CD4 T cell response toward the production of IFN-g interleukin-2 (IL-2) (Th1 phenotype). The balance between Th1 and Th2 CD4 T cell activation plays a critical role in determining whether or not to the autoimmune response generated will be destructive (see below).
There has been considerable speculation concerning factors responsible for the initiation of the disease process. Susceptibility to diabetes both in human and in the animal models is determined by the major histocompatibility complex (MHC) genotype; the disease-prone character is determined by genes in the class II region of the MHC complex (see Chapter 3). However, although susceptibility to the development of diabetes is under genetic control, the clinical disease itself is not solely determined by genotype. Thus, in the case of identical twins there is only a 30-40% concordance for disease. In the NOD mouse model, where all individuals are genetically identical as the results of inbreeding, disease develops in only 20-40% of male NOD animals in approximately 80% of females. The observation that the incidence of disease increases when animals are maintained under specific pathogenic-free conditions indicated an environmental contribution to the development of disease.10 The negative correlation with infection was an unexpected finding; one notion relating to the development of this disease was that the process resulted from a cross-reactive immune response to some environmental pathogen.11 If this were the case, one would expect the disease incidence to fall when animals were spared the immunological challenge of environmental pathogens.
Another interesting characteristic of the disease process is that although clinical disease is seen only in a proportion of animals in the NOD colony, all animals of the NOD genotype have pathology in their pancreas. That is, all animals express autoimmunity and show the development of lymphocytic infiltrates around islet tissue within the pancreas.12 There is, however, a difference between animals that develop clinical disease and those that have insulitis but do not go on to develop overt diabetes. In the latter case, the lymphocytes accumulate around the outside of the islet and rarely penetrate within the islet itself. In the former situation there is extensive invasion of islets by mononuclear cells and associated destruction of islet b-cells.12 It is worth noting that following destruction of islet b-cells the lymphocytic infiltrate disappears from the pancreas in one is left with small pseudo-islets made up predominantly of a-cells with little or no lymphocytic accumulation around the tissue. We must conclude therefore that the disease process is dependent on recognition of some b-cell-associated antigen that is not expressed by other cells within islet.
Studies carried out in transgenic animals where particular viral antigens are expressed in islet b-cells demonstrate that neither the expression of foreign antigens nor the possession of T cells specific for such antigens is a sufficient requirement for the precipitation of disease.13 The initiation of disease requires the "appropriate" presentation of antigen to an immune system that has potentially reactive T cells. We do not fully understand what is involved in "appropriate" antigens presentation. What we know is that infection of animals with a particular virus can lead to islet destruction when some viral antigens are expressed on islet b-cells. However, the development of a destructive process is not always seen in such situations. Variability appears to depend on the genetic background of the animal and the nature of the virus used in such studies.13
What is now becoming clear is that two distinct forms of autoimmune response can be observed. There is the response that leads to islet damage and the development of diabetes, which we can define as destructive autoimmunity. There is also a form of response that leads to pathology and associated lymphocyte accumulation around pancreatic islet but that is a nondestructive process and does not lead to clinical disease. The coexistence of the potential for either destructive or nondestructive autoimmunity in animals of a defined genotype suggests the development of diabetes is a stochastic process. That is, although we can precisely define the kinetics of the disease process and the proportion of animals in any group that will express overt diabetes at any given time (provided we are dealing with a colony maintained under controlled conditions), we are unable to specify beforehand exactly which animals will become diabetic. That is, we are unable to predict which animals will undergo the destructive process and which will lead to develop nondestructive autoimmunity before the process is well under way. Clearly, environmental conditions have a large influence on the proportion of animals that fall into either of these groups. This is why animals of the same genotype held under conventional laboratory conditions have a much lower incidence of disease than those maintained under specific pathogen-free conditions.
We do not precisely know what is responsible for the development of the disease process leading to clinical diabetes. We know that the genotype-specifically the class II MHC antigens type-in both animals and humans is a major factor controlling the disease-prone character. However, possession of the disease-prone genotype is not in itself sufficient requirement for the development of disease. Moreover, T cells with the capacity to recognize and respond to islet autoantigens can exist animals that do not develop clinical diabetes. All we can say with any confidence is that disease develops and disease-prone animals and individuals following "appropriate" stimulation of the immune system. The problem is to define what is meant by the term "appropriate stimulation."
Pathogenesis of the Disease Process
When it comes to understanding the basis of the pathogenic process involved in b-cell destruction in the development of IDDM we are on somewhat firmer ground. As we mentioned above, the disease process is a T cell-depended phenomenon in which both CD4 and CD8 T cells are required for the initiation of disease but where the CD4 T cell is the immunologically specific effector cell. CD4 T cells of the disease-prone individual are probably not interactions directly with antigen expressed on islet b-cells . Such antigens would be presented in association with class II MHC antigens; that is, in a form which does not favor CD4 T cell recognition. The CD4 T cells are most likely to be interacting with antigen derived from b-cells and processed by class II MHC-bearing antigen-presenting cells. This process would lead to the development and expression of cell-mediated immunity in which cytokine production by the inflammatory cells is a major pathogenic event leading to islet destruction.
There is evidence that free radical production is involved in this process. Both superoxide production by activation macrophages and nitric oxide production by islets in response to IL-1 appear to be involved in the pathogenic process (see Chapter 5). The Okamoto hypothesis provides the most useful model for understanding the involvement of three radical damage in the disease process (see Chapter 5). According to this hypothesis free radicals initiate DNA damage, which in turn activates the DNA repair enzyme (poly ADP-ribose synthetase) that is involved in DNA repair. This enzyme uses cellular NAD in the process and as a result depletes the b-cells' radical scavenging capacity. The cycling to this process results in b-cell death. One production of this model is that iron-chelating agents such as desferrioxamine, which block the conversion of super oxide to the more damaging hydroxyl radical, and specific inhibitors of the DNA repair enzyme such as nicotinamide, may be used to regulate this destructive process. Experimental evidence is consistent with such predictions.14
It has been suggested that nicotinamide is then agents that need be used to prevent the development of diabetes in a clinical situation. Certainly this agent is effective in animal models. However, the Okamoto hypothesis indicates that the effect of nicotinamide is to block the DNA repair enzyme. This process made lead to the development of oncogenic change because of interference with the DNA repair process. Earlier studies that examined the effect of nicotinamide on streptozotocin-induced diabetes indicated that nicotinamide could prevent the induction of diabetes by this agent.15 However, animals protected in this way were tumor prone and a significant proportion developed b-cell adenomas later in life. Any use of nicotinamide for the control of autoimmune diabetes in a clinical situation should be approached with considerable caution.
Exposure to environmental pathogens could have a marked effect on the development of diabetes in disease-prone animals. Environmental stimulation of the immune system leads to a decrease in the proportion of animals that go on to develop clinical disease. Immunostimulation of disease-prone animals with agents such as completes Freund's adjuvant or the vaccine BCG has been shown to block the development of diabetes in disease-prone NOD animals.16,17 Immunostimulation with either of these agents blocks the development of disease both when administered early, that is, soon after weaning, or quite late in the pathogenic process. Animals vaccinated at 85 days of age (clinical disease is first expressed from 90 days on) do not go on to develop clinical disease.17 Such immunostimulation does not inhibit the development of autoimmunity. It does, however, force the autoimmune response along the nondestructive pathway.17 Although such animals are protected from the development of overt diabetes they remain disease prone, and administration of cyclophosphamide can acutely result in the development of diabetes in these animals.17
These observations emphasize the need to view the immune system as an integrated network regulated by both positive and negative influences. Although "appropriate" stimulation of the immune system can lead to the development of diabetes in disease-prone animals, it is now also clear that other forms of immunostimulation can negatively regulate this process. This positive and negative regulation of immune function is correlated with the nature of cytokine production in either destructive or nondestructive lesions.12 In the former case, local sites within destructive lesions are producing IFN-g in high proportion and they contain relatively low numbers of IL-4-producing T cells (high proportion of Th1 cells). The nondestructive lesion, on the other hand, has a smaller proportion of IFN-g producing cells in the higher ratio of IL-4-producing cells (high proportion of Th2) cells. More direct evidence for cytokine involvement in this process comes from studies in which antibodies to IL-10 and IL-4 were used in an attempt to reverse the protective effect of immunostimulation. In the studies antibody treatment was shown to reverse the protective adjuvant effect (Calcinaro et al, unpublished data).
Although the mechanism of the adjuvant effect is not fully understood, the fact that quite marked effects have been obtained in animal models using both complete Freund's adjuvant and the more benign BCG vaccine has prompted clinical testing of BCG vaccination in newly diagnosed diabetic individuals. A preliminary clinical study of this kind has provided evidence that BCG vaccination may alter the natural history of the disease process in humans.17 These studies were carried out in an unblinded fashion and use historical controls and must, therefore, be considered as preliminary observations. Further studies carried out in a blinded fashion are required to establish whether or not BCG vaccination can alter the pathogenesis of diabetes in humans. If such studies are positive it may be possible to prevent the development of clinical disease in humans by immunostimulation as is the case in the experimental model.
The development of spontaneous diabetes as seen both in animals and humans is an autoimmune process dependent on the development of islet specific cell-mediated immunity. b-cell destruction is the result of inflammatory tissue damage and the specificity of this process appears to reflect the sensitivity of b-cells to radical damage. The class II MHC antigen genotype of both animals and humans regulate their disease-prone status. However, whether or not clinical disease develops in such individuals is the result of a balance between positive and negative regulation within the immune system. Such regulation is associated with differential cytokine production. Thus, appropriate stimulation of the immune system can lead either to the development of diabetes or to the development of nondestructive autoimmunity. This latter observation may provide the means for safe regulation of diabetes in disease-prone individuals.
Reference List - links to PubMed available in Reference List.
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