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.     

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.     

Role of calcium in pancreatic islet cell death by IFN-gamma/TNF-alpha

We studied the intracellular events associated with pancreatic beta cell apoptosis by IFN-gamma/TNF-alpha synergism. IFN-gamma/TNF-alpha treatment of MIN6N8 insulinoma cells increased the amplitude of high voltage-activated Ca(2+) currents, while treatment with IFN-gamma or TNF-alpha alone did not. Cytosolic Ca(2+) concentration ([Ca(2+)](c)) was also increased by IFN-gamma/TNF-alpha treatment. Blockade of L-type Ca(2+) channel by nifedipine abrogated death of insulinoma cells by IFN-gamma/TNF-alpha. Diazoxide that attenuates voltage-activated Ca(2+) currents inhibited MIN6N8 cell death by IFN-gamma/TNF-alpha, while glibenclamide that accentuates voltage-activated Ca(2+) currents augmented insulinoma cell death. A protein kinase C inhibitor attenuated MIN6N8 cell death and the increase in [Ca(2+)](c) by IFN-gamma/TNF-alpha. Following the increase in [Ca(2+)](c), calpain was activated, and calpain inhibitors decreased insulinoma cell death by IFN-gamma/TNF-alpha. As a downstream of calpain, calcineurin was activated and the inhibition of calcineurin activation by FK506 diminished insulinoma cell death by IFN-gamma/TNF-alpha. BAD phosphorylation was decreased by IFN-gamma/TNF-alpha because of the increased calcineurin activity, which was reversed by FK506. IFN-gamma/TNF-alpha induced cytochrome c translocation from mitochondria to cytoplasm and activation of caspase-9. Effector caspases such as caspase-3 or -7 were also activated by IFN-gamma/TNF-alpha treatment. These results indicate that IFN-gamma/TNF-alpha synergism induces pancreatic beta cell apoptosis by Ca(2+) channel activation followed by downstream intracellular events such as mitochondrial events and caspase activation and also suggest the therapeutic potential of Ca(2+) modulation in type 1 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.     

IFN-gamma sensitizes MIN6N8 insulinoma cells to TNF-alpha-induced apoptosis by inhibiting NF-kappaB-mediated XIAP upregulation

Although X-linked inhibitor of apoptosis protein (XIAP) is an important intracellular suppressor of apoptosis in a variety of cell types, its role in cytokine-induced pancreatic beta-cell apoptosis remains unclear. Here, we found that: (i) XIAP level was inversely correlated with tumor necrosis factor (TNF)-alpha-induced apoptosis in MIN6N8 insulinoma cells; (ii) adenoviral XIAP overexpression abrogated the TNF-alpha-induced apoptosis through inhibition of caspase activity; (iii) downregulation of XIAP by antisense oligonucleotide or Smac peptide sensitized MIN6N8 cells to TNF-alpha-induced apoptosis; (iv) XIAP expression was induced by TNF-alpha through a nuclear factor-kappaB (NF-kappaB)-dependent pathway, and interferon (IFN)-gamma prevented such an induction in a manner independent of NF-kappaB, which presents a potential mechanism underlying cytotoxic IFN-gamma/TNF-alpha synergism. Taken together, our results suggest that XIAP is an important modulator of TNF-alpha-induced apoptosis of MIN6N8 cells, and XIAP regulation in pancreatic beta-cells might play an important role in pancreatic beta-cell apoptosis and in the pathogenesis of type 1 diabetes.     

IL-12 administration accelerates autoimmune diabetes in both wild-type and IFN-gamma-deficient nonobese diabetic mice,revealing pathogenic and protective effects of IL-12-induced IFN-gamma

IL-12 administration to nonobese diabetic (NOD) mice induces IFN-gamma-secreting type 1 T cells and high circulating IFN-gamma levels and accelerates insulin-dependent diabetes mellitus (IDDM). Here we show that IL-12-induced IFN-gamma production is dispensable for diabetes acceleration, because exogenous IL-12 could enhance IDDM development in IFN-gamma-deficient as well as in IFN-gamma-sufficient NOD mice. Both in IFN-gamma(+/-) and IFN-gamma(-/-) NOD mice, IL-12 administration generates a massive and destructive insulitis characterized by T cells, macrophages, and CD11c(+) dendritic cells, and increases the number of pancreatic CD4(+) cells secreting IL-2 and TNF-alpha. Surprisingly, IL-12-induced IFN-gamma hinders pancreatic B cell infiltration and inhibits the capacity of APCs to activate T cells. Although pancreatic CD4(+) T cells from IL-12-treated IFN-gamma(-/-) mice fail to up-regulate the P-selectin ligand, suggesting that their entry into the pancreas may be impaired, T cell expansion is favored in these mice compared with IL-12-treated IFN-gamma(+/-) mice because IL-12 administration in the absence of IFN-gamma leads to enhanced cell proliferation and reduced T cell apoptosis. NO, an effector molecule in beta cell destruction, is produced ex vivo in high quantity by pancreas-infiltrating cells through a mechanism involving IL-12-induced IFN-gamma. Conversely, in IL-12-treated IFN-gamma-deficient mice, other pathways of beta cell death appear to be increased, as indicated by the up-regulated expression of Fas ligand on Th1 cells in the absence of IFN-gamma. These data demonstrate that IFN-gamma has a dual role, pathogenic and protective, in IDDM development, and its deletion allows IL-12 to establish alternative pathways leading to diabetes acceleration.     

The pro-apoptotic BH3-only protein Bid is dispensable for development of insulitis and diabetes in the non-obese diabetic mouse

Type 1 diabetes is caused by death of insulin-producing pancreatic beta cells. Beta-cell apoptosis induced by FasL may be important in type 1 diabetes in humans and in the non-obese diabetic (NOD) mouse model. Deficiency of the pro-apoptotic BH3-only molecule Bid protects beta cells from FasL-induced apoptosis in vitro. We aimed to test the requirement for Bid, and the significance of Bid-dependent FasL-induced beta-cell apoptosis in type 1 diabetes. We backcrossed Bid-deficient mice, produced by homologous recombination and thus without transgene overexpression, onto a NOD genetic background. Genome-wide single nucleotide polymorphism analysis demonstrated that diabetes-related genetic regions were NOD genotype. Transferred beta cell antigen-specific CD8+ T cells proliferated normally in the pancreatic lymph nodes of Bid-deficient mice. Moreover, Bid-deficient NOD mice developed type 1 diabetes and insulitis similarly to wild-type NOD mice. Our data indicate that beta-cell apoptosis in type 1 diabetes can proceed without Fas-induced killing mediated by the BH3-only protein Bid.     

Perturbations in nuclear factor-kappaB or c-Jun N-terminal kinase pathways in pancreatic beta cells confer susceptibility to cytokine-induced cell death

Pro-inflammatory cytokines have been implicated in the death of pancreatic beta cells leading to type 1 diabetes. NIT-1 cells are an insulinoma cell line derived from mice expressing the SV40 large T antigen. These cells are a useful tool in analysis of beta cell death. NIT-1 cells are highly susceptible to caspase-dependent apoptosis induced by TNF-alpha alone. Primary islets are not susceptible to cell death induced by TNF-alpha alone; however, they are killed by TNF-alpha and IFN-gamma in a nitric oxide-dependent manner. We examined signal transduction in NIT-1 cells in response to cytokines to determine the mechanism for TNF-alpha-induced apoptosis. We found that NIT-1 cells are defective in the activation of nuclear factor-kappaB (NFkappaB) as a result of functionally deficient RelA activity, because overexpression of RelA protected NIT-1 cells from apoptosis. TNF-alpha also did not induce phosphorylation of c-Jun N-terminal kinase in NIT-1 cells. Together, these defects prevent expression of anti-apoptotic genes in NIT-1 cells and make them susceptible to TNF-alpha. To determine whether similar defects in primary beta cells would induce the same effect, we examined TNF-alpha-induced apoptosis in islets isolated from mice deficient in NFkappaB p50. These islets were as susceptible as wild-type islets to TNF-alpha and IFN-gamma-induced cell death. In contrast to wild-type islets, cell death was not prevented by inhibition of nitric oxide in p50-deficient islets. Blocking NFkappaB has been proposed as a mechanism for protection of beta cells from cytokine-induced cell death in vivo. Our results suggest that this would make beta cells equally or more sensitive to cytokines.     

TNF receptor 1-dependent beta cell toxicity as an effector pathway in autoimmune diabetes

Autoimmune diabetes is characterized by a chronic progressive inflammatory autoimmune reaction that ultimately causes the selective elimination of pancreatic beta cells. To address the question of whether the cell death-inducing cytokines TNF and lymphotoxin alpha are involved in this process, we generated nonobese diabetic (NOD) mice that are deficient for TNF receptor 1 (TNFR1 or TNFRp55). Insulitis developed in these mice similarly to that in normal control NOD mice, but progression to diabetes was completely abrogated. Since this was probably due to the complex immunomodulatory effects of TNF and lymphotoxin alpha signaled via TNFR1 on lymphohemopoietic cells, adoptive transfer experiments with spleen cells from diabetic NOD mice were conducted. It was found that the absence of TNFR1 in recipients delayed diabetes induced by normal control and precluded diabetes induced by perforin-deficient spleen cells. In a CD8+ T cell-mediated model of diabetes, however, diabetes induced by adoptive transfer of TCR transgenic lymphocytic choriomeningitis virus glycoprotein-specific CD8+ T cells was not delayed by the absence of TNFR1 in recipient mice. Together with the described expression patterns of perforin and TNF in the mononuclear islet infiltrates of NOD mice, these results indicate that two diabetogenic effector mechanisms are delivered by distinct cell populations: CD8+ T cells lyse beta cells via perforin-dependent cytotoxicity, whereas CD4+ T cells, macrophages, and dendritic cells contribute to diabetes development via TNFR1-dependent beta cell toxicity.     

Paralytic autoimmune myositis develops in nonobese diabetic mice made Th1 cytokine-deficient by expression of an IFN-gamma receptor beta-chain transgene

Nonobese diabetic (NOD) mice and some human type 1 diabetes (T1D) patients manifest low to high levels of other autoimmune pathologies. Skewing their cytokine production from a Th1 (primarily IFN-gamma) to a Th2 (primarily IL-4 and IL-10) pattern is a widely proposed approach to dampen the pathogenicity of autoreactive diabetogenic T cells. However, it is important that altered cytokine balances not enhance any other autoimmune proclivities to dangerous levels. Murine CD4 T cells are characterized by a reciprocal relationship between the production of IFN-gamma and expression of the beta-chain component of its receptor (IFN-gamma RB). Thus, NOD mice constitutively expressing a CD2 promoter-driven IFN-gamma RB transgene in all T cells are Th1-deficient. Unexpectedly, NOD.IFN-gamma RB Tg mice were found to develop a lethal early paralytic syndrome induced by a CD8 T cell-dependent autoimmune-mediated myositis. Furthermore, pancreatic insulitis levels were not diminished in 9-wk-old NOD.IFN-gamma RB Tg females, and overt T1D developed in the few that survived to an older age. Autoimmune-mediated myositis is only occasionally detected in standard NOD mice. Hence, some manipulations diminishing Th1 responses can bring to the forefront what are normally secondary autoimmune pathologies in NOD mice, while also failing to dependably abrogate pancreatic beta cell destruction. This should raise a cautionary note when considering the use of protocols that induce alterations in cytokine balances as a means of blocking progression to overt T1D in at-risk humans.     

Beta-cell apoptosis in an accelerated model of autoimmune diabetes.

BACKGROUND: The non-obese diabetic (NOD) mouse is a model of human type 1 diabetes in which autoreactive T cells mediate destruction of pancreatic islet beta cells. Although known to be triggered by cytotoxic T cells, apoptosis has not been unequivocally localized to beta cells in spontaneously diabetic NOD mice. We created a model of accelerated beta-cell destruction mediated by T cells from spontaneously diabetic NOD mice to facilitate the direct detection of apoptosis in beta cells. MATERIALS AND METHODS: NOD.scid (severe combined immunodeficiency) mice were crossed with bm1 mice transgenically expressing the costimulatory molecule B7-1 (CD80) in their beta cells, to generate B7-1 NOD.scid mice. Apoptosis in islet cells was measured as DNA strand breakage by the TdT-mediated-dUTP-nick end labeling (TUNEL) technique. RESULTS: Adoptive transfer of splenocytes from spontaneously diabetic NOD mice into B7-1 NOD.scid mice caused diabetes in recipients within 12-16 days. Mononuclear cell infiltration and apoptosis were significantly greater in the islets of B7-1 NOD.scid mice than in nontransgenic NOD.scid mice. Dual immunolabeling for TUNEL and either B-7 or insulin, or the T cell markers CD4 and CD8, and colocalization by confocal microscopy clearly demonstrated apoptosis in beta cells as well in a relatively larger number of infiltrating T cells. The clearance time of apoptotic beta cells was estimated to be less than 6 min. CONCLUSIONS: B7-1 transgenic beta cells undergo apoptosis during their accelerated destruction in response to NOD mouse effector T cells. Rapid clearance implies that beta cells undergoing apoptosis would be detected only rarely during more protracted disease in spontaneously diabetic NOD mice.     

Prevention of autoimmune diabetes in NOD mice by troglitazone is associated with modulation of ICAM-1 expression on pancreatic islet cells and IFN-gamma expression in splenic T cells

Thiazolidinediones acting as PPAR-gamma agonists are a new generation of oral antidiabetics addressing insulin resistance as a main feature of type-2 diabetes. In accordance to our results, pre-clinical studies have demonstrated that the thiazolinedione troglitazone prevents the development of insulin-dependent autoimmune type-1 diabetes. To investigate whether TGZ acts by affecting the ICAM-1/LFA-1 pathway and/or the Th1/Th2 cytokine balance in NOD mice, we analysed the IL-1beta-induced ICAM-1 expression on islet-cells and the LFA-1, CD25, IL-2, IFN-gamma, IL-4, and IL-10 expression on splenocytes. After 200 days of oral TGZ administration, islet cells from TGZ-treated NOD mice showed a reduced ICAM-1 expression in response to the pro-inflammatory cytokine IL-1beta. The expression of the ligand LFA-1 on CD4(+) and CD8(+) T-cells was comparable to that of placebo- and untreated controls. Also, the expression of Th1/Th2 cytokines was comparable in groups receiving TGZ or Placebo. Nevertheless, the investigated NOD mice segregated into IFN-gamma low- and IFN-gamma high producers as revealed by cluster analysis. Interestingly, the majority of TGZ-treated mice belonged to the cluster of IFN-gamma low producers. Thus, the prevention of autoimmune diabetes in NOD mice by TGZ seems to be associated with suppression of IL-1beta-induced ICAM-1 expression leading to a reduced vulnerability of pancreatic beta-cells during the effector stage of beta-cell destruction. In addition, IFN-gamma production was modulated, implicating that alteration of the Th1/Th2 cytokine balance might have contributed to diabetes prevention. The findings of this study suggest that TGZ exerts its effects by influencing both the beta-cells as the target of autoimmune beta-cell destruction and the T-cells as major effectors of the autoimmune process.     

IFN-gamma affects homing of diabetogenic T cells.

IFN-gamma is a cytokine with pleiotropic functions that participates in immune and autoimmune responses. The lack of IFN-gamma is known to delay the development of autoimmune diabetes in nonobese diabetic (NOD) mice. Splenocytes from diabetic NOD and IFN-gamma knockout (KO) NOD mice transfer diabetes into NOD recipients equally well. However, adoptive transfer of diabetogenic T cells from NOD mice into NOD.IFN-gamma-KO or NOD mice lacking beta-chain of IFN-gamma receptor (NOD.IFN-gammaRbeta-KO) appeared to be much less efficient. We found that IFN-gamma influences the ability of diabetogenic cells to penetrate pancreatic islets. Tracing in vivo of insulin-specific CD8+ T cells has shown that homing of these cells to the islets of Langerhans was affected by the lack of IFN-gamma. While adhesion of insulin-specific CD8+ cells to microvasculature was normal, the diapedesis was significantly impaired. This effect was reversible by treatment of the animals with rIFN-gamma. Thus, IFN-gamma may, among other effects, influence immune and autoimmune responses by supporting the homing of activated T cells.     

Ganglioside GM1 effects on the expression of nerve growth factor (NGF), Trk-A receptor, proinflammatory cytokines and on autoimmune diabetes onset in non-obese diabetic (NOD) mice

NOD (non-obese diabetic) mice develop type 1 diabetes mellitus spontaneously and with a strong similarity to the human disease. Differentiation and function of pancreas beta cells are regulated by a variety of hormones and growth factors, including the nerve growth factor (NGF). Gangliosides have multiple immunomodulatory activities with immunosuppressive properties, decreasing lymphoproliferative responses and modulating cytokine production. In the present study, serum, pancreas islets and spleen mononuclear cells from NOD mice treated with monosialic ganglioside GM1 (100 mg/kg/day) and the group control which received saline solution were isolated to investigate the proinflammatory cytokines (IL-1beta, IFN-gamma, IL-12, TNF-alpha), NGF and its high-affinity receptor TrkA, peri-islet Schwann cells components (GFAP, S100-beta) expression and the relationship with diabetes onset and morphological aspects. Our results suggest that GM1 administration to female NOD mice beginning at the 4th week of life is able to reduce the index of inflammatory infiltrate and consequently the expression of diabetes, modulating the expression of proinflammatory cytokines (IL-12, IFN-gamma, TNF-alpha and IL-1beta). Furthermore, GM1 increases GFAP, S-100beta and NGF in pancreas islets, factors involved in beta cell survival.     

Molecular mechanisms for gender differences in susceptibility to T cell-mediated autoimmune diabetes in nonobese diabetic mice

Nonobese diabetic (NOD) mice spontaneously develop diabetes with a strong female prevalence; however, the mechanisms for this gender difference in susceptibility to T cell-mediated autoimmune diabetes are poorly understood. This investigation was initiated to find mechanisms by which sex hormones might affect the development of autoimmune diabetes in NOD mice. We examined the expression of IFN-gamma, a characteristic Th1 cytokine, and IL-4, a characteristic Th2 cytokine, in islet infiltrates of female and male NOD mice at various ages. We found that the most significant difference in cytokine production between sexes was during the early stages of insulitis at 4 wk of age. IFN-gamma was significantly higher in young females, whereas IL-4 was higher in young males. CD4(+) T cells isolated from lymph nodes of female mice and activated with anti-CD3 and anti-CD28 Abs produced more IFN-gamma, but less IL-4, as compared with males. Treatment of CD4(+) T cells with estrogen significantly increased, whereas testosterone treatment decreased the IL-12-induced production of IFN-gamma. We then examined whether the change in IL-12-induced IFN-gamma production by treatment with sex hormones was due to the regulation of STAT4 activation. We found that estrogen treatment increased the phosphorylation of STAT4 in IL-12-stimulated T cells. We conclude that the increased susceptibility of female NOD mice to the development of autoimmune diabetes could be due to the enhancement of the Th1 immune response through the increase of IL-12-induced STAT4 activation by estrogen.     

A mechanism for IL-10-mediated diabetes in the nonobese diabetic (NOD) mouse: ICAM-1 deficiency blocks accelerated diabetes

Neonatal islet-specific expression of IL-10 in nonobese diabetic (NOD) mice accelerates the onset of diabetes, whereas systemic treatment of young NOD mice with IL-10 prevents diabetes. The mechanism for acceleration of diabetes in IL-10-NOD mice is not known. Here we show, by adoptive transfers, that prediabetic or diabetic NOD splenocytes upon encountering IL-10 in the pancreatic islets readily promoted diabetes. This outcome suggests that the compartment of exposure, not the timing, confers proinflammatory effects on this molecule. Moreover, injection of IL-10-deficient NOD splenocytes into transgenic IL-10-NOD.scid/scid mice elicited accelerated disease, demonstrating that pancreatic IL-10 but not endogenous IL-10 is sufficient for the acceleration of diabetes. Immunohistochemical analysis revealed hyperexpression of ICAM-1 on the vascular endothelium of IL-10-NOD mice. The finding suggests that IL-10 may promote diabetes via an ICAM-1-dependent pathway. We found that introduction of ICAM-1 deficiency into IL-10-NOD mice as well as into NOD mice prevented accelerated insulitis and diabetes. Failure to develop insulitis and diabetes was preceded by the absence of GAD65-specific T cell responses. The data suggest that ICAM-1 plays a role in the formation of the "immunological synapse", thereby affecting the generation and/or expansion of islet-specific T cells. In addition, ICAM-1 also played a role in the effector phase of autoimmune diabetes because adoptive transfer of diabetogenic BDC2.5 T cells failed to elicit clinical disease in ICAM-1-deficient IL-10-NOD and NOD mice. These findings provide evidence that pancreatic IL-10 is sufficient to drive pathogenic autoimmune responses and accelerates diabetes via an ICAM-1-dependent pathway.