Growth factor-induced signaling of the pancreatic epithelium.

Activated signaling proteins regulate diverse processes, including the differentiation of the pancreatic islet cells during ontogeny. Here we uncover the in vivo phosphorylation status of major growth factor-activated signaling proteins in normal adult mice and during pancreatic islet regeneration. We report elevated phospho-mitogen-activated protein kinase (phospho-MAPK), phospho-c-Jun-NH2-terminal kinase (phospho-JNK), and phospho-p38 MAPK expression during pancreatic regeneration. Immunoblotting experiments demonstrated elevated phosphorylation of p52 Src-homology/collagen (SHC) in the ductal network as well, substantiating the activation of this pathway. Furthermore, protein kinase B (PKB/Akt), a key signaling protein in the anti-apoptotic pathway, was phosphorylated to a greater extent in the ductal network from regenerating pancreas. We observed fibroblast growht factor (FGF)10 and platelet-derived growth factor (PDGF)AA expression in embryonic as well as regenerating adult pancreas. Epidermal growth factor (EGF) and PDGFAA stimulated MAPK and Akt phosphorylation, while FGF10 stimulated MAPK but not Akt phosphorylation in a time-dependent manner in freshly isolated cells from the adult ductal network. These data suggest that a heightened level of expression and stimulation of key signaling proteins underlie the expansion and differentiation processes that support pancreatic ontogeny and regeneration.

Report from the 1st International NOD Mouse T-Cell Workshop and the follow-up mini-workshop.

A workshop on autoreactive T-cell responses in NOD mice was held to optimize autoreactive T-cell detection methodologies. Using different proliferation assay protocols, 1 of the 11 participating laboratories detected spontaneous T-cell responses to GAD(524-543) and insulin(9-23) in their NOD mice. Two other laboratories were able to detect autoreactive responses when using enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA) analysis of cytokines in culture supernatants, suggesting that these assays provided greater sensitivity. To address the divergent findings, a follow-up mini-workshop tested NOD mice from four different colonies side-by-side for T-cell proliferative responses to an expanded panel of autoantigens, using the protocol that had enabled detection of responses in the 1st International NOD Mouse T-Cell Workshop. Under these assay conditions, 16 of 16 NOD mice displayed proliferative responses to whole GAD65, 13 of 16 to GAD(524-543), 9 of 16 to GAD(217-236), 7 of 16 to insulin(9-23), and 5 of 16 to HSP277. Thus, spontaneous proliferative T-cell responses can be consistently detected to some beta-cell autoantigens and peptides thereof. Overall, the results suggest that more sensitive assays (e.g., ELISPOT, ELISA analysis of cytokines in supernatants, or tetramer staining) may be preferred for the detection of autoreactive T-cells.

TNF-alpha receptor 1 (p55) on islets is necessary for the expression of LIGHT on diabetogenic T cells.

Insulin-dependent diabetes mellitus results from T-cell-mediated destruction of pancreatic islet beta cells. Both CD4 and CD8 T cells have been shown to be independently capable of beta cell destruction. However, the mechanism of beta cell destruction has remained elusive. It has previously been shown that the absence of TNF-alpha receptor 1 (p55) on the islets protected islets from CD4 T-cell-mediated destruction as long as the T cells did not have access to wild-type islets in vivo. Wild-type and TNF-alpha receptor 1 (p55) deficient islets induce similar levels of proliferation of BDC2.5 T cells. In this study, we demonstrate that islet TNF-alpha receptor 1 (p55) influences the expression of LIGHT (TNFSF-14), a TNF family member with both cytolytic and costimulatory properties, on BDC2.5 T cells and the expression of its receptor HVEM (TNFRSF-14) by islets, indicating a role for LIGHT-HVEM interactions in autoimmune diabetes.

IL-4 triggers autoimmune diabetes by increasing self-antigen presentation within the pancreatic Islets.

Several findings have recently questioned the long held hypothesis that cytokines belonging to the Th2 pathway are protective in T-cell-mediated autoimmunity. Among them, there is our previous report that pancreatic expression of IL-4 activated islet antigen-specific BDC2.5 T cells and rendered them able to trigger insulin-dependent diabetes mellitus in ins-IL-4/BDC2.5 mice (Mueller et al., Immunity, 7, 1997). Here we analyze the mechanisms underlying IL-4-mediated activation of the self-reactive BDC2.5 T cells. IL-4 is mainly known as the Th2-driving cytokine. However, IL-4 is also critical for DC maturation and upregulation of antigen uptake and presentation by macrophages. In our model, we found that pancreatic expression of IL-4 activated self-reactive BDC2.5 T cells by increasing islet antigen presentation by macrophages and dendritic cells. IL-4 could have triggered self-antigen presentation within the pancreatic islets both by driving maturation of DC from a tolerizing to a priming state and by increasing self-antigen uptake by macrophages.

Interleukin-4 acts at the locus of the antigen-presenting dendritic cell to counter-regulate cytotoxic CD8+ T-cell responses.

The mechanism underlying suppression of immune responses by interleukin-4 (IL-4) has remained unexplained. Here we show that the antigen-presenting dendritic cell is central to counter-regulation of autoimmune disease by IL-4. IL-4 acts at the locus of the dendritic cell to decrease the cytolytic T-cell response, preventing autoimmunity. Stimulation of cytotoxic precursors by antigen pulsed dendritic cells induces their differentiation but the process is blocked by IL-4. IL-4-influenced DC produce distinct effects on CD8+ T cells depending on their state of activation. The molecular basis for this regulation is the alteration of the expression ratio of the costimulatory ligands B7.1/B7.2 on dendritic cells. Our findings demonstrate that B7.2 induces expansion of CD8+ T cells and B7.1 governs their acquisition of cytolytic activity. IL-4 influences the dendritic cell to elicit qualitative differences in T-cell responses, providing the basis for counter-regulation mediated by IL-4.

IL-18 directs autoreactive T cells and promotes autodestruction in the central nervous system via induction of IFN-gamma by NK cells.

IL-18 promotes NK cell and Th1 cell activity and may bridge innate and adaptive immune responses. Myelin oligodendrocyte glycoprotein (MOG) is a myelin component of the CNS and is a candidate autoantigen in multiple sclerosis. In the present study we show that IL-18-deficient (IL-18-/-) mice are defective in mounting autoreactive Th1 and autoantibody responses and are resistant to MOG35-55 peptide-induced autoimmune encephalomyelitis. IL-18 administration enhances the disease severity in wild-type mice and restores the ability to generate Th1 response in the IL-18-/- mice. This restoration was abrogated in NK cell-depleted mice, indicating that the action of IL-18 in promoting the generation of MOG-specific Th cells was dependent on NK cells. Furthermore, transfer of NK cells from recombinase-activating gene 1-/- mice, but not from recombinase-activating gene 1/IFN-gamma-/- mice, rescued the defective Th1 responses in IL-18-/- mice and rendered IL-18-/- mice susceptible to the induction of autoimmune encephalomyelitis. Thus, IL-18 can direct autoreactive T cells and promote autodestruction in the CNS at least in part via induction of IFN-gamma by NK cells.

Islet-specific expression of IL-10 promotes diabetes in nonobese diabetic mice independent of Fas, perforin, TNF receptor-1, and TNF receptor-2 molecules.

Several death-signaling or death-inducing molecules have been implicated in beta cell destruction, including Fas, perforin, and TNFR-1. In this study, we examined the role of each death-signaling molecule in the IL-10-accelerated diabetes of nonobese diabetic (NOD) mice. Groups of IL-10-NOD mice, each deficient in either Fas, perforin, or TNFR-1 molecules, readily developed insulitis, and subsequently succumbed to diabetes with an accelerated kinetics and incidence similar to that observed in their wild-type or heterozygous IL-10-NOD littermates. Similarly, a TNFR-2 deficiency did not block accelerated diabetes in IL-10-NOD mice and spontaneous diabetes in NOD mice. These results demonstrate that pancreatic IL-10 promotes diabetes independent of Fas, perforin, TNFR-1, and TNFR-2 molecules. Subsequently, when cyclophosphamide, a diabetes-inducing agent, was injected into insulitis-free NOD. lpr/lpr mice, none of these mice developed insulitis or diabetes. Our data suggest that cyclophosphamide- but not IL-10-induced diabetes is Fas dependent. Overall, these findings provide evidence that pancreatic expression of IL-10 promotes diabetes independent of the major death pathways and provide impetus for identification of novel death pathways precipitating autoimmune destruction of insulin-producing beta cells.

Pancreatic expression of interferon-gamma protects mice from lethal coxsackievirus B3 infection and subsequent myocarditis.

Cardiovascular disease is one of the leading causes of death worldwide, and has been associated with many environmental risk factors. Recent evidence has indicated the involvement of pathogens such as viruses as causative agents, and specifically identified the coxsackievirus B serogroup as the leading culprit. Not only has coxsackievirus B3 (CB3) been identified from patients with cardiovascular disease, but also infection of mice with CB3 strains can reproduce human clinical heart disease in rodents. Several mechanisms have been proposed in an attempt to distinguish between pathology mediated by direct viral destruction of cardiac muscle cells or by the virus-induced immune response directed at infected myocytes or at 'mimicked' epitopes shared between viral and cardiac antigens. To distinguish between these mechanisms, we infected a unique mouse that diminishes the extent of infection and spread of the virus, but allows complete immunity to the virus. Transgenic mice expressing interferon-gamma in their pancreatic beta cells failed to develop CB-3-induced myocarditis. This work challenges the idea of the function of the immune response and 'molecular mimicry' in the CB-3-induced autoimmune myocarditis model, and instead favors the idea of virus-mediated damage. These results emphasize the benefit of reducing the level of viremia early during infection, thereby reducing the incidence of virus-mediated heart damage and autoimmunity.

Expression of ErbB receptors during pancreatic islet development and regrowth.

We have characterized expression of the ErbB receptor family and one of its ligands, heregulin, in an effort to identify molecules associated with pancreatic development and regeneration. In addition to studying expression during fetal pancreatic development, we have also studied expression during pancreatic regeneration in the interferon-gamma (IFNgamma)-transgenic mouse, which exhibits significant duct cell proliferation and new islet formation. These studies demonstrate significant expression of the ErbB2, ErbB3, and ErbB4 receptors, in addition to heregulin isoforms, in the developing murine fetal pancreas. We also report significant ductal expression of these proteins during IFNgamma-mediated pancreatic regeneration. This striking expression was absent in 1-week-old neonates, but was clearly visible in pups by 5 weeks of age. These data therefore indicate that ErbB receptor and ligand expression decline by birth in both the IFNbeta-transgenic and non-transgenic mice, and that expression resumes early in postnatal life in the IFNbeta-transgenic mice. The expression of ErbB receptor family members at sites of islet development and regrowth suggests that these molecules might be relevant to these processes.

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.

Necrotizing myopathy induced by overexpression of interferon-gamma in transgenic mice.

A transgenic mouse model has been established in which the cytokine interferon-gamma (IFN-gamma) is overexpressed through the action of the acetylcholine receptor epsilon promoter acting at the neuromuscular junction. While originally developed as a model for the study of the pathogenesis of myasthenia gravis, there are important differences from both human myasthenia gravis and its animal model, experimental autoimmune myasthenia gravis. By 4 months of age there was a well-established inflammatory, predominantly necrotizing myopathy, with marked dystrophic calcification. Dystrophic and degenerative changes in terminal axons and adjacent Schwann cells were also apparent. The acetylcholine receptor was not the primary target of the inflammatory response, since at 10 weeks of age the receptor content was not decreased and antibodies were not detected bound to the receptor. The IFNgamma transgenic mouse model may provide a clinically relevant model of necrotizing myopathy for investigation of the pathological changes associated with, and presumably precipitated by, overexpression of the pro-inflammatory cytokine interferon-gamma on the neuromuscular junction, intramuscular nerves and myofibers.

The Th2 cytokine IL-4 is not required for the progression of antibody-dependent autoimmune myasthenia gravis.

Experimental autoimmune myasthenia gravis (EAMG), a disorder of the neuromuscular junction, is mediated by autoantibodies against muscle nicotinic acetylcholine receptor (AChR). The roles of IFN-gamma (Th1) and IL-4 (Th2) cytokines in the initiation and progression of this disease are not fully understood. Recently, we have demonstrated that IFN-gamma is necessary for the initiation of tAChR-induced EAMG in mice. However, the role of IL-4 in the progression of clinical EAMG remained undetermined. In this study we have addressed the contribution of IL-4 in the disease progression in IL-4(-/-) C57BL/6j mice whose IL-4 gene has been disrupted. Following immunization with Torpedo (t) AChR, the IL-4(-/-) mice readily developed signs of muscle weakness and succumbed to clinical EAMG with kinetics similar to the susceptibility of IL-4(+/+) mice. The tAChR-primed lymph node cells from IL-4(-/-) mice vigorously proliferated to tAChR and to its dominant alpha146-162 sequence associated with disease pathogenesis. However, these T cells secreted higher levels of IFN-gamma and IL-2, suggesting the development of a Th1 default pathway in these mice. Nevertheless, the IL-4 mutation had no effect on the recruitment of CD4+ Vbeta6+ T cells specific to the dominant tAChR alpha146-162 sequence in vivo. Immune sera from IL-4(-/-) mice showed a dramatic increase in mouse AChR-specific IgG2a levels followed by a concomitant decrease in IgG1 levels, but these mice did not exhibit an accelerated disease. In conclusion, we have demonstrated for the first time that IL-4 is not required either for the generation of a pathogenic anti-AChR humoral immune response or for progression of clinical EAMG in mice.

Cellular immune response to adenoviral vector infected cells does not require de novo viral gene expression: implications for gene therapy.

Replication-defective adenoviral (RDAd) vectors can be generated at high titers and infect both dividing and nondividing cells. Long term expression in the transduced tissue, however, has been a problem because of the cellular immune responses against the infected cells. We demonstrate that mice injected with RDAd vectors containing mouse leptin gene reduce food intake and lose weight for only 7 to 10 days. Splenocytes obtained from infected mice are able to lyse target cells infected with RDAd vectors. Surprisingly, target cells infected with psoralen-treated, UV-crosslinked, biologically inactive RDAd also were lysed efficiently by the effector cells. Furthermore, splenocytes obtained from mice injected with inactive RDAd vectors efficiently lysed target cells infected with RDAd vectors. Whether RDAd vectors were injected i.m. or i.v. or through an i.p. route, the extent of lysis was similar. We propose that cells infected with RDAd vectors present antigens for recognition by class 1 major histocompatibility complex-restricted cytotoxic T lymphocytes by a mechanism that does not require viral replication or de novo protein synthesis. These results should prompt reevaluation of the use of RDAd vectors for gene therapy when long-term expression is required.

Cytokines and IDDM: implications for etiology and therapy.

The evidence that human insulin-dependent diabetes mellitus (IDDM) is a T cell-mediated disease is well substantiated, and the use of transgenic technology to understand the Th1/Th2 paradigm will provide keys to attenuating pathogenic autoimmunity. Insofar as the role of Th1 cytokines in IDDM is concerned, interferon gamma is considered a critical player in the etiology, a proinflammatory role has been determined for IDDM, interleukin-2 is considered an "amplification" factor, and tumor necrosis factor-alpha presents dichotomous effects. Regarding the role of Th2 cytokines in IDDM, interleukin-4 is essential for immunoprotection and counterregulation of IDDM, and interleukin-10 plays immunoprotective and destructive roles. Therefore, Th1 and Th2 cytokines, when expressed individually in islets of Langerhans, have provided surprising results in manipulating the IDDM of transgenic NOD mice. The current data show that the same cytokine can produce either protective or pathological effects, depending upon the timing of its participation in the disease process. Of all the cytokines examined, IL-4 seems to be the likely candidate for preventing IDDM.

Lentivirus-mediated transduction of islet grafts with interleukin 4 results in sustained gene expression and protection from insulitis.

Autoimmune destruction of islets in the pancreas leads to the development of insulin-dependent diabetes mellitus (IDDM). Replacement of insulin-producing tissue by transplantation of islets provides a cure to disease but requires immunosuppression or a means of controlling anti-graft immune responses. To promote islet survival we have utilized a local approach by expressing immunoregulatory molecules in islet grafts. The results presented here show that the human immunodeficiency virus (HIV)-based lentiviral vector is capable of stably transducing whole islets. Foreign reporter gene expression was observed both in vitro and in vivo 30 days after transplantation. Grafts containing insulin-positive beta-islet cells expressing foreign protein indicate that transduction does not interfere with glucose regulation. The absence of inflammatory infiltrates in grafts suggests that transduction does not activate the immune system. When islets transduced with an HIV vector expressing IL-4 were transplanted into diabetes-prone mice, animals were protected from autoimmune insulitis and islet destruction. As demonstrated by proliferative and cytokine analysis, protection was consistent with a switching of islet-antigen-specific T cell responses toward a Th2 phenotype. These results suggest that HIV-based lentivirus vectors can efficiently transduce islet cells with genes encoding potentially therapeutic molecules, for possibly managing diabetes.

Interferon-gamma protects against herpes simplex virus type 1-mediated neuronal death.

Host inflammatory mediators, such as interferons, play a protective role in infection, but the mechanism is undefined and may differ between tissue compartments. To determine whether interferon-gamma (IFN-gamma) elicitation prevents destructive encephalitis in herpes simplex virus type 1 (HSV-1) infection of the central nervous system, IFN-gamma-knockout (GKO) mice were challenged intravitreally with HSV-1 strain F, inciting infection of the eyes and the brain. Indeed, the GKO mice showed encephalitis with ataxia, whereas nontransgenic controls remained asymptomatic. Morphology and digoxigenin labeling of DNA fragments revealed increased apoptosis in the brains of GKO mice compared with controls, although viral replication was not influenced at early stages of infection. Greater numbers of apoptotic cells in the brains of GKO mice correlated with neurological symptoms, as well as lower expression of the protective protooncogene bcl-2. Thus, IFN-gamma inhibits apoptosis, affording neuronal protection from destructive encephalitis during viral infection of the central nervous system.

CD40 ligand-CD40 interactions are necessary for the initiation of insulitis and diabetes in nonobese diabetic mice.

The nonobese diabetic (NOD) mouse spontaneously develops T cell-dependent autoimmune diabetes. Here, we investigate the role of CD40 ligand (CD40L)-CD40 costimulation in the initiation and progression of this disease. Anti-CD40L mAb treatment of 3- to 4-wk-old NOD females (the age at which insulitis typically begins) completely prevented the insulitis and diabetes. In contrast, treatment of such mice with anti-CD40L at >9 wk of age did not inhibit the disease process. These results suggest that a costimulatory signal by CD40L is required early but not in the effector phase of disease development. Anti-CD40L treatment affected the priming of islet Ag-specific T cell responses in vivo. Cytokine analysis revealed a dramatic decrease in IFN-gamma and IL-2 release without a concomitant increase in IL-4 production by T cells from anti-CD40L-treated mice. Thus, anti-CD40L impaired the islet Ag-specific Th1 cell response in vivo, and the prevention of diabetes by anti-CD40L was not associated with switching of the response from a Th1 to a Th2 profile. Cotransfer of splenocytes from anti-CD40L-treated mice with splenocytes from diabetic NOD mice into NOD/scid mice did not inhibit the transfer of disease, indicating that anti-CD40L does not prevent the disease by inducing regulatory cells. Since anti-CD40L clearly prevented the insulitis by inhibiting the development and further accumulation of pathogenic Th1 cells to islets of Langerhans, we conclude that CD40L-CD40 costimulation is required for early events in the development of spontaneous autoimmune diabetes.

Interferon gamma (IFN-gamma) is necessary for the genesis of acetylcholine receptor-induced clinical experimental autoimmune myasthenia gravis in mice.

Experimental autoimmune myasthenia gravis (EAMG) is an animal model of human myasthenia gravis (MG). In mice, EAMG is induced by immunization with Torpedo californica acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA). However, the role of cytokines in the pathogenesis of EAMG is not clear. Because EAMG is an antibody-mediated disease, it is of the prevailing notion that Th2 but not Th1 cytokines play a role in the pathogenesis of this disease. To test the hypothesis that the Th1 cytokine, interferon (IFN)-gamma, plays a role in the development of EAMG, we immunized IFN-gamma knockout (IFN-gko) (-/-) mice and wild-type (WT) (+/+) mice of H-2(b) haplotype with AChR in CFA. We observed that AChR-primed lymph node cells from IFN-gko mice proliferated normally to AChR and to its dominant pathogenic alpha146-162 sequence when compared with these cells from the WT mice. However, the IFN-gko mice had no signs of muscle weakness and remained resistant to clinical EAMG at a time when the WT mice exhibited severe muscle weakness and some died. The resistance of IFN-gko mice was associated with greatly reduced levels of circulating anti-AChR antibody levels compared with those in the WT mice. Comparatively, immune sera from IFN-gko mice showed a dramatic reduction in mouse AChR-specific IgG1 and IgG2a antibodies. However, keyhole limpet hemocyanin (KLH)-priming of IFN-gko mice readily elicited both T cell and antibody responses, suggesting that IFN-gamma regulates the humoral immune response distinctly to self (AChR) versus foreign (KLH) antigens. We conclude that IFN-gamma is required for the generation of a pathogenic anti-AChR humoral immune response and for conferring susceptibility of mice to clinical EAMG.

Local delivery of interleukin 4 by retrovirus-transduced T lymphocytes ameliorates experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory autoimmune disease of the central nervous system which serves as a model for the human disease multiple sclerosis. We demonstrate here that encephalitogenic T cells, transduced with a retroviral gene, construct to express interleukin 4, and can delay the onset and reduce the severity of EAE when adoptively transferred to myelin basic protein-immunized mice. Thus, T lymphocytes transduced with retroviral vectors can deliver "regulatory cytokines" in a site-specific manner and may represent a viable therapeutic strategy for the treatment of autoimmune disease.