Significant role for Fas in the pathogenesis of autoimmune diabetes

Programmed cell death represents an important pathogenic mechanism in various autoimmune diseases. Type I diabetes mellitus (IDDM) is a T cell-dependent autoimmune disease resulting in selective destruction of the beta cells of the islets of Langerhans. beta cell apoptosis has been associated with IDDM onset in both animal models and newly diagnosed diabetic patients. Several apoptotic pathways have been implicated in beta cell destruction, including Fas, perforin, and TNF-alpha. Evidence for Fas-mediated lysis of beta cells in the pathogenesis of IDDM in nonobese diabetic (NOD) mice includes: 1) Fas-deficient NOD mice bearing the lpr mutation (NOD-lpr/lpr) fail to develop IDDM; 2) transgenic expression of Fas ligand (FasL) on beta cells in NOD mice may result in accelerated IDDM; and 3) irradiated NOD-lpr/lpr mice are resistant to adoptive transfer of diabetes by cells from NOD mice. However, the interpretation of these results is complicated by the abnormal immune phenotype of NOD-lpr/lpr mice. Here we present novel evidence for the role of Fas/FasL interactions in the progression of NOD diabetes using two newly derived mouse strains. We show that NOD mice heterozygous for the FasL mutation gld, which have reduced functional FasL expression on T cells but no lymphadenopathy, fail to develop IDDM. Further, we show that NOD-lpr/lpr mice bearing the scid mutation (NOD-lpr/lpr-scid/scid), which eliminates the enhanced FasL-mediated lytic activity induced by Fas deficiency, still have delayed onset and reduced incidence of IDDM after adoptive transfer of diabetogenic NOD spleen cells. These results provide evidence that Fas/FasL-mediated programmed cell death plays a significant role in the pathogenesis of autoimmune diabetes.     

Evidence that beta cell death in the nonobese diabetic mouse is Fas independent.

Recent studies suggest that Fas expression on pancreatic beta cells may be important in the development of autoimmune diabetes in the nonobese diabetic (NOD) mouse. To address this, pancreatic islets from NOD mice were analyzed by flow cytometry to directly identify which cells express Fas and Fas ligand (FasL) ex vivo and after in vitro culture with cytokines. Fas expression was not detected on beta cells isolated from young (35 days) NOD mice. In vitro, incubation of NOD mouse islets with both IL-1 and IFN-gamma was required to achieve sufficient Fas expression and sensitivity for islets to be susceptible to lysis by soluble FasL. In islets isolated from older (>/=125 days) NOD mice, Fas expression was detected on a limited number of beta cells (1-5%). FasL was not detected on beta cells from either NOD or Fas-deficient MRLlpr/lpr islets. Also, both NOD and MRLlpr/lpr islets were equally susceptible to cytokine-induced cell death. This eliminates the possibility that cytokine-treated murine islet cells commit "suicide" due to simultaneous expression of Fas and FasL. Last, we show that NO is not required for cytokine-induced Fas expression and Fas-mediated apoptosis of islet cells. These findings indicate that beta cells can be killed by Fas-dependent cytotoxicity; however, our results raise further doubts about the clinical significance of Fas-mediated beta cell destruction because few Fas-positive cells were isolated immediately before the development of diabetes.     

Mechanisms of accelerated immune-mediated diabetes resulting from islet beta cell expression of a Fas ligand transgene

Nonobese diabetic (NOD) mice transgenic for Fas ligand (FasL) on islet beta cells (HIPFasL mice) exhibit an accelerated diabetes distinct from the normal autoimmune diabetes of NOD mice. This study was undertaken to define the mechanism underlying accelerated diabetes development in HIPFasL mice. It was found that diabetes in HIPFasL mice is dependent on the NOD genetic background, as HIPFasL does not cause diabetes when crossed into other mice strains and is lymphocyte dependent, as it does not develop in HIPFasL(SCID) mice. Diabetes development in NOD(SCID) recipients of diabetic HIPFasL splenocytes is slower than when using splenocytes from diabetic NOD mice. Beta cells from HIPFasL mice are more susceptible to cytokine-induced apoptosis than wild-type NOD beta cells, and this can be blocked with anti-FasL Ab. HIPFasL islets are more rapidly destroyed than wild-type islets when transplanted into nondiabetic NOD mice. This confirms that FasL(+) islets do not obtain immune privilege, and instead NOD beta cells constitutively expressing FasL are more susceptible to apoptosis induced by Fas-FasL interaction. These findings are consistent with the accelerated diabetes of young HIPFasL mice being a different disease process from the autoimmune diabetes of wild-type NOD mice. The data support a mechanism by which cytokines produced by the insulitis lesion mediate up-regulation of beta cell Fas expression, resulting in suicide or fratricide of HIPFasL beta cells that overexpress FasL.     

Comparing the relative role of perforin/granzyme versus Fas/Fas ligand cytotoxic pathways in CD8+ T cell-mediated insulin-dependent diabetes mellitus.

CD8+ cytotoxic T cells play a critical role in initiating insulin-dependent diabetes mellitus. The relative contribution of each of the major cytotoxic pathways, perforin/granzyme and Fas/Fas ligand (FasL), in the induction of autoimmune diabetes remains controversial. To evaluate the role of each lytic pathway in beta cell lysis and induction of diabetes, we have used a transgenic mouse model in which beta cells expressing the influenza virus hemagglutinin (HA) are destroyed by HA-specific CD8+ T cells from clone-4 TCR-transgenic mice. Upon adoptive transfer of CD8+ T cells from perforin-deficient clone-4 TCR mice, there was a 30-fold increase in the number of T cells required to induce diabetes. In contrast, elimination of the Fas/FasL pathway of cytotoxicity had little consequence. When both pathways of cytolysis were eliminated, mice did not become diabetic. Using a model of spontaneous diabetes, which occurs in double transgenic neonates that express both clone-4 TCR and Ins-HA transgenes, mice deficient in either the perforin or FasL/Fas lytic pathway become diabetic soon after birth. This indicates that, in the neonate, large numbers of autoreactive CD8+ T cells can lead to destruction of islet beta cells by either pathway.     

Crosstalk between keratinocytes and T lymphocytes via Fas/Fas ligand interaction: modulation by cytokines.

Apoptosis mediated by Fas/FasL interaction plays an important role during many inflammatory skin disorders. To estimate whether the expression of FasL, the ligand for Fas, might be regulated by cytokines we stimulated primary human keratinocytes with several pro- and anti-inflammatory cytokines. Keratinocytes cultured to subconfluence expressed FasL constitutively. Cells stimulated with the proinflammatory cytokines IL-1beta, TNF-alpha, IFN-gamma, and IL-15, respectively, increased significantly their intracellular as well as cell surface-bound FasL expression in a time- and dose-dependent manner. This cytokine-induced FasL expression was dependent on new protein synthesis. Despite enhanced expression of cell surface-bound FasL, no release of soluble FasL was measured in the cell supernatants determined by ELISA. Stimulation of the cells with IL-6, IL-10, IL-12, TGF-beta1, and GM-CSF did not modulate the constitutive FasL expression, but IFN-gamma-mediated FasL up-regulation was significantly diminished by IL-10 and TGF-beta1, respectively. Up-regulation of FasL on IFN-gamma-stimulated keratinocytes led to increased apoptosis within monolayers cultured for 48 h. Moreover, coculture experiments performed with Fas+ Jurkat T cells revealed that enhanced FasL expression on IFN-gamma-stimulated keratinocytes induced apoptosis in cocultured T cells, demonstrating that up-regulated FasL was functionally active. In summary, our data suggest the important regulatory role of cytokine-controlled Fas/FasL interaction in the cross-talk between keratinocytes and skin-infiltrating T cells for maintenance of homeostasis in inflammatory skin processes.     

Mutation in the Fas pathway impairs CD8+ T cell memory

Fas death pathway is important for lymphocyte homeostasis, but the role of Fas pathway in T cell memory development is not clear. We show that whereas the expansion and contraction of CD8+ T cell response against Listeria monocytogenes were similar for wild-type (WT) and Fas ligand (FasL) mutant mice, the majority of memory CD8+ T cells in FasL mutant mice displayed an effector memory phenotype in the long-term in comparison with the mainly central memory phenotype displayed by memory CD8+ T cells in WT mice. Memory CD8+ T cells in FasL mutant mice expressed reduced levels of IFN-gamma and displayed poor homeostatic and Ag-induced proliferation. Impairment in CD8+ T cell memory in FasL mutant hosts was not due to defective programming or the expression of mutant FasL on CD8+ T cells, but was caused by perturbed cytokine environment in FasL mutant mice. Although adoptively transferred WT memory CD8+ T cells mediated protection against L. monocytogenes in either the WT or FasL mutant hosts, FasL mutant memory CD8+ T cells failed to mediate protection even in WT hosts. Thus, in individuals with mutation in Fas pathway, impairment in the function of the memory CD8+ T cells may increase their susceptibility to recurrent/latent infections.     

Fas and Fas ligand in embryos and adult mice: ligand expression in several immune-privileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover

The cell surface receptor Fas (FasR, Apo-1, CD95) and its ligand (FasL) are mediators of apoptosis that have been shown to be implicated in the peripheral deletion of autoimmune cells, activation-induced T cell death, and one of the two major cytolytic pathways mediated by CD8+ cytolytic T cells. To gain further understanding of the Fas system., we have analyzed Fas and FasL expression during mouse development and in adult tissues. In developing mouse embryos, from 16.5 d onwards, Fas mRNA is detectable in distinct cell types of the developing sinus, thymus, lung, and liver, whereas FasL expression is restricted to submaxillary gland epithelial cells and the developing nervous system. Significant Fas and FasL expression were observed in several nonlymphoid cell types during embryogenesis, and generally Fas and FasL expression were not localized to characteristic sites of programmed cell death. In the adult mouse, RNase protection analysis revealed very wide expression of both Fas and FasL. Several tissues, including the thymus, lung, spleen, small intestine, large intestine, seminal vesicle, prostate, and uterus, clearly coexpress the two genes. Most tissues constitutively coexpressing Fas and FasL in the adult mouse are characterized by apoptotic cell turnover, and many of those expressing FasL are known to be immune privileged. It may be, therefore, that the Fas system is implicated in both the regulation of physiological cell turnover and the protection of particular tissues against potential lymphocyte-mediated damage.     

Hypothesis: A recurrent, moderate activation fosters systemic autoimmunity — the apoptotic roles of TCR, IL-2 and fas ligand

Studies of several gene knockout mice suggest an interesting association of a moderate T cell response with systemic autoimmune diseases. In addition, CD95 ligand (FasL) expression in some strains of these mice is impaired. Because FasL is critically involved in regulating peripheral tolerance, there may be a link between autoimmune diseases and a moderate T cell response that cannot activate the FasL gene. Here, we propose that there are two thresholds of T cell activation. When moderately stimulated, T cells can be activated to the low (1st) threshold, which permits the induction of CD40L, IL-2, IL-4, and other components that help the immune response. The high (2nd) activation threshold can only be achieved by a strong and concurrent stimulation through TCR and IL-2R. Once the high threshold is reached, FasL is produced to induce apoptosis of the activated T and B cells. In the absence of the FasL-mediated downregulation, the activated B cells become efficient antigen-presenting cells for self-antigens and excellent responders for T cell help. Such an exacerbating condition, induced by recurrent and moderate activation, favors the development of autoreactive T cells and autoantibody production. Evidence supporting this hypothesis and some predictions that can be tested are described.     

Constitutive activation of the Fas ligand gene in mouse lymphoproliferative disorders.

Mice homozygous for lpr (lymphoproliferation) or gld (generalized lymphoproliferative disease) develop lymphadenopathy and splenomegaly and suffer from autoimmune disease. The lpr mice have a defect in a cell-surface receptor, Fas, that mediates apoptosis, while gld mice have a mutation in the Fas ligand (FasL). Northern hybridization with the FasL cDNA as probe indicated that the cells accumulating in lpr and gld mice abundantly express the FasL mRNA without stimulation. By means of in situ hybridization and immunohistochemistry, we identified the cells expressing the FasL mRNA as CD4-CD8- double negative T cells. The T cells from lpr mice were specifically cytotoxic against Fas-expressing cells. Since FasL is normally expressed in activated mature T cells these results indicate that the double negative T cells accumulating in lpr and gld mice are activated once, and support the notion that the Fas/FasL system is involved in activation-induced suicide of T cells. Furthermore, the graft-versus host disease caused by transfer of lpr bone marrow to wild-type mice can be explained by the constitutive expression of the FasL in lpr-derived T cells.     

IFN-gamma/TNF-alpha synergism as the final effector in autoimmune diabetes: a key role for STAT1/IFN regulatory factor-1 pathway in pancreatic beta cell death

Fas ligand (FasL), perforin, TNF-alpha, IL-1, and NO have been considered as effector molecule(s) leading to beta cell death in autoimmune diabetes. However, the real culprit(s) in beta cell destruction have long been elusive, despite intense investigation. We and others have demonstrated that FasL is not a major effector molecule in autoimmune diabetes, and previous inability to transfer diabetes to Fas-deficient nonobese diabetic (NOD)-lpr mice was due to constitutive FasL expression on lymphocytes from these mice. Here, we identified IFN-gamma/TNF-alpha synergism as the final effector molecules in autoimmune diabetes of NOD mice. A combination of IFN-gamma and TNF-alpha, but neither cytokine alone, induced classical caspase-dependent apoptosis in insulinoma and pancreatic islet cells. IFN-gamma treatment conferred susceptibility to TNF-alpha-induced apoptosis on otherwise resistant insulinoma cells by STAT1 activation followed by IFN regulatory factor (IRF)-1 induction. IRF-1 played a central role in IFN-gamma/TNF-alpha-induced cytotoxicity because inhibition of IRF-1 induction by antisense oligonucleotides blocked IFN-gamma/TNF-alpha-induced cytotoxicity, and transfection of IRF-1 rendered insulinoma cells susceptible to TNF-alpha-induced cytotoxicity. STAT1 and IRF-1 were expressed in pancreatic islets of diabetic NOD mice and colocalized with apoptotic cells. Moreover, anti-TNF-alpha Ab inhibited the development of diabetes after adoptive transfer. Taken together, our results indicate that IFN-gamma/TNF-alpha synergism is responsible for autoimmune diabetes in vivo as well as beta cell apoptosis in vitro and suggest a novel signal transduction in IFN-gamma/TNF-alpha synergism that may have relevance in other autoimmune diseases and synergistic anti-tumor effects of the two cytokines.     

Fibronectin-associated Fas ligand rapidly induces opposing and time-dependent effects on the activation and apoptosis of T cells

Recently, it has been shown that Fas ligand (FasL) interacts with the extracellular matrix (ECM) protein fibronectin (FN), and that the bound FasL retains its cytotoxic efficacy. Herein, we examined the ramifications of FasL-ECM protein interactions throughout a specific time period, in the absence or presence of additional activating molecules, assuming that these complexed interactions occur during inflammation. We found that exposure of purified human T cells to FN-associated recombinant FasL for as brief as 5-10 min at 0.1-100 ng/ml induced their adhesion in beta(1) integrin- and FasR-dependent manners while activating the intracellular protein kinase, Pyk-2. The FN-associated FasL stops the CXCL12 (stromal cell-derived factor 1alpha)-induced chemotaxis of T cells by inhibiting the chemokine-induced extracellular signal-regulated kinase signaling and cytoskeletal rearrangement. This short term exposure of T cells to the FN-bound FasL (1 ng/ml), which was followed by T cell activation via the CD3 complex, resulted in 1) increased secretion of IFN-gamma (measured after 24 h), and 2) enhanced T cell apoptosis (measured after 72 h). Thus, in the context of inflamed ECM and depending on the time after FasL activation, its concentration, and the nature of other contextual mediators, FasL initially retains effector T cells at sites of inflammation and, later, induces T cell apoptosis and return to homeostasis.     

Inhibition of autoimmune diabetes by Fas ligand: the paradox is solved

Previous reports that diabetogenic lymphocytes did not induce diabetes in nonobese diabetic (NOD)-lpr mice suggested the critical role of Fas-Fas ligand (FasL) interaction in pancreatic beta cell apoptosis. However, recent works demonstrated that FasL is not an effector molecule in islet beta cell death. We addressed why diabetes cannot be transferred to NOD-lpr mice despite the nonessential role of Fas in beta cell apoptosis. Lymphocytes from NOD-lpr mice were constitutively expressing FasL. A decrease in the number of FasL+ lymphocytes by neonatal thymectomy facilitated the development of insulitis. Cotransfer of FasL+ lymphocytes from NOD-lpr mice completely abrogated diabetes after adoptive transfer of lymphocytes from diabetic NOD mice. The inhibition of diabetes by cotransferred lymphocytes was reversed by anti-FasL Ab, indicating that FasL on abnormal lymphocytes from NOD-lpr mice was responsible for the inhibition of diabetes transfer. Pretreatment of lymphocytes with soluble FasL (sFasL) also inhibited diabetes transfer. sFasL treatment decreased the number of CD4+CD45RBlow cells and increased the number of propidium iodide-stained cells among CD4+CD45RBlow cells, suggesting that sFasL induces apoptosis on CD4+CD45RBlow "memory" cells. These results resolve the paradox between previous findings and suggest a new role for FasL in the treatment of autoimmune disorders. Our data also suggest that sFasL is involved in the deletion of potentially hazardous peripheral "memory" cells, contrary to previous reports that Fas on unmanipulated peripheral lymphocytes is nonfunctional.     

Identification of a CD8 T cell that can independently mediate autoimmune diabetes development in the complete absence of CD4 T cell helper functions

Previous work has indicated that an important component for the initiation of autoimmune insulin-dependent diabetes mellitus (IDDM) in the NOD mouse model entails MHC class I-restricted CD8 T cell responses against pancreatic beta cell Ags. However, unless previously activated in vitro, such CD8 T cells have previously been thought to require helper functions provided by MHC class II-restricted CD4 T cells to exert their full diabetogenic effects. In this study, we show that IDDM development is greatly accelerated in a stock of NOD mice expressing TCR transgenes derived from a MHC class I-restricted CD8 T cell clone (designated AI4) previously found to contribute to the earliest preclinical stages of pancreatic beta cell destruction. Importantly, these TCR transgenic NOD mice (designated NOD.AI4alphabeta Tg) continued to develop IDDM at a greatly accelerated rate when residual CD4 helper T cells were eliminated by introduction of the scid mutation or a functionally inactivated CD4 allele. In a previously described stock of NOD mice expressing TCR transgenes derived from another MHC class I-restricted beta cell autoreactive T cell clone, IDDM development was retarded by elimination of residual CD4 T cells. Hence, there is variability in the helper dependence of CD8 T cells contributing to the development of autoimmune IDDM. The AI4 clonotype represents the first CD8 T cell with a demonstrated ability to progress from a naive to functionally activated state and rapidly mediate autoimmune IDDM development in the complete absence of CD4 T cell helper functions.     

Th17 cells undergo Fas-mediated activation-induced cell death independent of IFN-gamma

IL-17-secreting CD4+ T cells (Th17 cells) play a critical role in immune responses to certain infections and in the development of many autoimmune disorders. The mechanisms controlling homeostasis in this cell population are largely unknown. In this study, we show that murine Th17 cells undergo rapid apoptosis in vitro upon restimulation through the TCR. This activation-induced cell death (AICD), a common mechanism for elimination of activated T cells, required the Fas and FasL interaction: Fas was stably expressed, while FasL was up-regulated upon TCR reactivation of Th17 cells; Ab ligation of Fas induced Th17 cell death; and AICD was completely absent in Th17 cells differentiated from gld/gld CD4+ T cells. Thus, the Fas/FasL pathway is essential in regulating the AICD of Th17 cells. Interestingly, IFN-gamma, a cytokine previously found to be important for the AICD of T cells, did not affect Th17 cell apoptosis. Furthermore, Th17 cells derived from mice deficient in IFN-gamma receptor 1 (IFN-gammaR1-/-) underwent AICD similar to wild-type cells. Thus, AICD of Th17 cells occurs via the Fas pathway, but is independent of IFN-gamma.     

Cell death induced by the Fas/Fas ligand pathway and its role in pathology.

Engagement of the cell death surface receptor Fas by Fas ligand (FasL) results in apoptotic cell death, mediated by caspase activation. Cell death mediated via Fas/FasL interaction is important for homeostasis of cells in the immune system and for maintaining immune-privileged sites in the body. Killing via the Fas/FasL pathway also constitutes an important pathway of killing for cytotoxic T cells. Fas ligand is induced in activated T cells, resulting in activation-induced cell death by the Fas/FasL pathway. Recently it has been shown that the Fas receptor can also be up-regulated following a lesion to the cell, particularly that induced by DNA-damaging agents. This can then result in killing of the cell by a Fas/FasL-dependent pathway. Up-regulation of Fas receptor following DNA damage appears to be p53 dependent.     

Possible role of organ-specific autoantigen for Fas ligand-mediated activation-induced cell death in murine Sj?gren's syndrome.

Activation-induced cell death (AICD) is a well-known mechanism of peripheral T cell tolerance that depends upon an interaction between Fas and Fas ligand (FasL). In this study, we demonstrate that the administration of a soluble form of anti-FasL Ab, FLIM58, results in severe destructive autoimmune exocrinopathy in the murine model of human Sj?gren's syndrome (SS), and we found that an organ-specific autoantigen may play an important role on down-modulation of AICD. A high titer of serum autoantibodies against 120-kDa alpha-fodrin autoantigen was detected in the FLIM58-treated mice, and splenic T cell culture supernatants contained high levels of IFN-gamma. In vitro T cell apoptosis assay indicated that FasL-mediated AICD is down-regulated by autoantigen stimulation in spleen cells from the murine SS model, but not from Fas-deficient MRL/lpr mice and FasL-deficient MRL/gld mice. FasL undergo metalloproteinase-mediated proteolytic processing in their extracellular domains, resulting in the release of soluble trimeric ligands (soluble FasL). We showed that the processing of soluble FasL occurs in autoantigen-specific CD4(+) T cells, and that a significant increase in expressions of metalloproteinase-9 mRNA was observed in spleen cells from SS model mice. These findings indicate that the increased generation of soluble FasL inhibits the normal AICD process, leading to the proliferation of effector CD4(+) T cells in the murine SS model.     

The human melanoma cell line MelJuSo secretes bioactive FasL and APO2L/TRAIL on the surface of microvesicles. Possible contribution to tumor counterattack

Tumor cells have developed multiple mechanisms to evade control by the immune system. Tumoral cells expressing Fas ligand (FasL) have been proposed to "counterattack" against activated antitumoral effector immune cells, although some authors have indicated that FasL is not expressed on the surface of the same tumors, such in the case of melanoma cells. However, other factors could be implicated, such as the balance of soluble versus membrane-bound forms or the secretion of death ligands on the surface of microvesicles, as described previously by our group in human T cells. In the present study, we analyzed the expression and secretion of FasL and APO2 ligand (APO2L)/TRAIL in the human melanoma cell line MelJuSo. We have observed the expression of preformed FasL and APO2L/TRAIL in these cells, their secretion associated with microvesicles upon melanoma activation with PHA or with alpha-melanocyte stimulating hormone (alpha-MSH), and the toxicity of these microvesicles against normal human T cell blasts. We have also observed that the mechanism of secretion of FasL and APO2L/TRAIL from melanoma cells is depending both on microtubules and actin filaments. From these data, it can be concluded that the MelJuSo melanoma cell line has the possibility to "counterattack" against activated immune effector cells. However, the in vivo outcome seems more complex since it has been also described that FasL expressed in tumors has a proinflammatory effect.     

Fas and Fas ligand immunolocalization in pancreatic islets of NOD mice during spontaneous and cyclophosphamide-accelerated diabetes

During insulin-dependent diabetes mellitus, immune cells which infiltrate pancreatic islets mediate beta cell destruction over a prolonged asymptomatic prediabetic period. The molecular mechanisms of beta cell death in vivo remain unresolved. At least two major molecular processes of destruction have been proposed. One involves the Fas–FasL (Fas–Fas ligand) system and the other, the perforin pathway. Here, dual-label immunohistochemistry was employed to examine the intra-islet expression, distribution and cellular sources of Fas and FasL in the NOD mouse, during spontaneous diabetes (days 21, 40 and 90) and following acceleration of diabetes with cyclophosphamide (days 0, 4, 7, 11 and 14 after cyclophosphamide administration). The expression of the proteins was correlated with advancing disease. FasL was expressed constitutively in most beta cells but not in glucagon or somatostatin cells or islet inflammatory cells and paralleled the loss of insulin immunolabelling with advancing disease. It was also expressed in beta cells of non-diabetes prone CD-1 and C57BL/6 mice from a young age (day 21). Strong immunolabelling for Fas was first observed in extra-islet macrophages and those close to the islet in NOD and non-diabetes-prone mice. During spontaneous and cyclophosphamide diabetes, it was observed in a higher proportion of islet infiltrating macrophages than CD4 and CD8 T cells, concomitant with advancing insulitis. In cyclophosphamide-treated mice, the proportion of Fas-positive intra-islet CD4 and CD8 T cells at day 14 (with and without diabetes) was considerably higher than at days 0, 4, 7 and 11. At days 11 and 14, a proportion of Fas-positive intra-islet macrophages co-expressed interleukin-1 and inducible nitric oxide synthase. Fas was not detectable in beta cells and other islet endocrine cells during spontaneous and cyclophosphamide induced diabetes. Our results show constitutive expression of FasL in beta cells in the NOD mouse and predominant expression of Fas in intra-islet macrophages and to a lesser extent in T cells prior to diabetes onset. Interleukin-1 in intra-islet macrophages may induce Fas and inducible nitric oxide synthase expression in an autocrine and paracrine manner and mediate beta cell destruction or even death of some macrophages and T cells. However, other mechanisms of beta cell destruction during spontaneous and cyclophosphamide-accelerated diabetes and independent of Fas–FasL, require examination.     

Diabetes and tolerance in transgenic mice expressing class II MHC molecules in pancreatic beta cells

Insulin-dependent diabetes is caused by the loss of insulin-producing beta cells in pancreatic islets. It has been proposed that aberrant expression of Class II Major Histocompatibility Complex (MHC) molecules on beta cells stimulates an autoimmune attack against beta cell antigens. To test this hypothesis, we generated transgenic mice that express Class II MHC molecules (E alpha d/E beta b, or I-Eb) on beta cells. Diabetes was found in 100% of transgenic progeny from three expressing transgenic mouse lines, but without evidence for lymphocytic infiltrates. Furthermore, T lymphocytes appeared to be tolerant to the transgene I-Eb molecule, despite the absence of expression of I-Eb in the thymus or any other lymphoid tissue. The results suggest that novel expression of Class II MHC molecules on nonlymphoid cells is by itself insufficient to initiate autoimmune responses against tissue-specific antigens.