Cholecystokinin expression in the developing and regenerating pancreas and intestine.

In developmental terms, the endocrine system of neither the gut nor the pancreatic islets has been characterized fully. Little is known about the involvement of cholecystokinin (CCK), a gut hormone, involved in regulating the secretion of pancreatic hormones, and pancreatic growth. Here, we tracked CCK-expressing cells in the intestines and pancreata of normal mice (BALB/c), Non Obese Diabetic (NOD) mice and interferon (IFN)-gamma transgenic mice, which exhibit pancreatic regeneration, during embryonic development, the postnatal period and adulthood. We also questioned whether IFN-gamma influences the expression of CCK. The results from embryonic day 16 showed that all three strains had CCK in the acinar region of pancreata, and specifically in alpha cells that also expressed glucagon. However, in adulthood only BALB/c and NOD mice continued this pattern. By contrast, in IFN-gamma transgenic mice, CCK expression was suppressed from birth to 3 months of age in the pancreata but not intestines. However, by 5 months of age, CCK expression appeared in the regenerating pancreatic ductal region of IFN-gamma transgenic mice. In the intestine, CCK expression persisted from fetus to adulthood and was not influenced by IFN-gamma. Intestinal cells expressing CCK did not co-express glucagon, suggesting that these cells are phenotypically distinct from CCK-expressing cells in the pancreatic islets, and the effect of IFN-gamma on CCK varies depending upon the cytokine's specific microenvironment.

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.

Transcription factor expression during pancreatic islet regeneration.

Recent studies by a number of laboratories have identified transcription factors that are involved in pancreatic development. Indeed, marked abnormalities in pancreatic development result from deficiencies in these molecules, which include, among others, PDX-1, islet-1 (Isl-1), and Pax-6. These studies have prompted us to evaluate the expression of Isl-1 and Pax-6 in the pancreas of the interferon-gamma (IFNgamma) transgenic mouse, which exhibits new islet growth and expansion of ducts throughout the life of the animal. We have previously demonstrated that PDX-1 is strikingly expressed in the ducts of the IFNgamma transgenic mouse. This latter observation compelled us to examine expression of hepatocyte nuclear factor-3beta (HNF3beta), which mediates PDX-1 gene transcription, in the IFNgamma transgenic pancreas as well. As a result of these studies, we now demonstrate marked expression of these transcription factors in the pancreatic ducts of IFNgamma transgenic mice. These data suggest a role for these transcription factors during pancreatic regeneration in the IFNgamma transgenic mouse.

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.

PDX-1 and Msx-2 expression in the regenerating and developing pancreas.

We have observed pancreatic duct cell proliferation and islet regeneration in transgenic mice whose pancreata produce interferon gamma (IFNg mice). We have previously demonstrated that new islet cells derive from endocrine progenitor cells in the pancreatic ducts of this model. The current study was initiated to define these endocrine progenitor cells further and to identify novel markers associated with pancreatic regeneration. Importantly, we have found that PDX-1, a transcription factor required for insulin gene transcription as well as for pancreatic development during embryogenesis, is expressed in the duct cells of IFNg mice. This striking observation suggests an important role for PDX-1 in the marked regeneration observed in IFNg mice, paralleling its critical function during ontogeny. Also demonstrated was elevated expression of the homeobox-containing protein Msx-2 in the pancreata of fetal mice as well as in adult IFNg mice, identifying this molecule as a novel marker associated with pancreatic development and regeneration as well. The identification of PDX-1 and Msx in the ducts of the IFNg transgenic pancreas but not in the ducts of the non-transgenic pancreas suggests that these molecules are associated with endocrine precursor cells in the ducts of the IFNg transgenic mouse.

CD4+ CD45RB low-density cells from untreated mice prevent acute allograft rejection.

In the absence of therapy that suppresses the action of the immune system, the immune response to transplantation Ags results in rapid rejection of the transplant. The most successful mechanism so far described that achieves organ-specific immunological tolerance is that which controls peripheral tolerance to self-tissue. Until now, no similarities have been documented between the peripheral response to self-Ags and the response to transplantation Ags. CD4+ cells that express a high density of CD45RB (in the mouse) and CD45RC (in the rat) on their surface have been shown to cause a number of autoimmune disorders. In contrast, autoimmunity caused by the CD45RB high-density cells is inhibited by CD4+ CD45RB cells that express a low density of CD45RB (CD45RC in the rat). In this paper we show that CD4+ CD45RB high-density cells are sufficient to cause rejection of a MHC-mismatched pancreas allograft, whereas CD4+ CD45RB low-density cells are not. Unexpectedly, the CD45RB low-density cells prevent the CD45RBhigh expressing cells from causing rejection. These data suggest that the response to foreign tissue can be controlled in the same way as the response to self-tissue.

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.

Transgenic expression of epidermal growth factor and keratinocyte growth factor in beta-cells results in substantial morphological changes.

The upregulation of a limited number of growth factors in our interferon-gamma transgenic model for regeneration within the pancreas lead us to propose that these factors are important during pancreatic regeneration. In this study, we have assessed the influence of two growth factors within the pancreas, epidermal growth factor (EGF) and keratinocyte growth factor (KGF), by ectopically expressing these proteins under the control of the human insulin promoter in transgenic mice. This beta-cell-targeted expression of either EGF or KGF resulted in significant morphological changes, including cellular proliferation and disorganized islet growth. Intercrossing the individual Ins-EGF and Ins-KGF transgenic mice resulted in more profound changes in pancreatic morphology including proliferation of pancreatic cells and extensive intra-islet fibrosis. Insulin-producing beta-cells were found in some of the ducts of older Ins-EGF and Ins-EGFxKGF transgenic mice, and amylase-producing cells were observed within the islet structures of the double transgenic mice. These data suggest that both EGF and KGF are capable of affecting pancreatic differentiation and growth, and that co-expression of these molecules in islets has a more substantial impact on the pancreas than does expression of either growth factor alone.

Interleukin-4 secretion by the allograft fails to affect the allograft-specific interleukin-4 response in vitro.

BACKGROUND: The role of the cytokine, interleukin (IL)-4, in allograft rejection and protection is not clear. We have previously shown that IL-4 transgenically expressed in a pancreas allograft does not protect the allograft from rejection. Here, we analyze the effect of the transgenically expressed IL-4 on the cytokine profile of the allograft-specific immune response. METHODS: C57BL/6SCID mice were infused with small numbers of spleen cells from C57BL/6 donors. The former received pancreas grafts from 1- to 2-day-old BALB/c donors which did or did not transgenically express IL-4 in the graft. Three weeks after the cell infusion, the spleens were removed and the splenocytes were restimulated in vitro with BALB/c APC, and third party BALB.K APC. IL-2 and IL-4 levels in the culture supernatants were measured. RESULTS: The presence of a pancreatic allograft induced an increase in the levels of both IL-2 and IL-4 in culture supernatants from splenocytes of mice receiving grafts compared with mice not receiving grafts. The presence of IL-4 transgenically expressed in the pancreas allograft had no effect on the in vitro cytokine profile. CONCLUSIONS: from these results we conclude that the failure of transgenically expressed IL-4 to protect the allograft was not associated with up-regulation of a graft antigen-specific IL-4 response.

Pancreatic expression of keratinocyte growth factor leads to differentiation of islet hepatocytes and proliferation of duct cells.

Keratinocyte growth factor, (KGF), a member of the fibroblast growth factor (FGF) family, is involved in wound healing. It also promotes the differentiation of many epithelial tissues and proliferation of epithelial cells as well as pancreatic duct cells. Additionally, many members of the highly homologous FGF family (including KGF), influence both growth and cellular morphology in the developing embryo. We have previously observed elevated levels of KGF in our interferon-gamma transgenic mouse model of pancreatic regeneration. To understand the role of KGF in pancreatic differentiation, we generated insulin promoter-regulated KGF transgenic mice. Remarkably, we have found that ectopic KGF expression resulted in the emergence of hepatocytes within the islets of Langerhans in the pancreas. Additionally, significant intra-islet duct cell proliferation in the pancreata of transgenic KGF mice was observed. The unexpected appearance of hepatocytes and proliferation of intra-islet duct cells in the pancreata of these mice evidently stemmed directly from local exposure to KGF.

Islet-specific Th1, but not Th2, cells secrete multiple chemokines and promote rapid induction of autoimmune diabetes.

Migration of CD4 cells into the pancreas represents a hallmark event in the development of insulin-dependent diabetes mellitus. Th1, but not Th2, cells are associated with pathogenesis leading to destruction of islet beta-cells and disease onset. Lymphocyte extravasation from blood into tissue is regulated by multiple adhesion receptor/counter-receptor pairs and chemokines. To identify events that regulate entry of CD4 cells into the pancreas, we transferred Th1 or Th2 cells induced in vitro from islet-specific TCR transgenic CD4 cells into immunodeficient (NOD.scid) recipients. Although both subsets infiltrated the pancreas and elicited multiple adhesion receptors (peripheral lymph node addressin, mucosal addressin cell adhesion molecule-1, LFA-1, ICAM-1, and VCAM-1) on vascular endothelium, entry/accumulation of Th1 cells was more rapid than that of Th2 cells, and only Th1 cells induced diabetes. In vitro, Th1 cells were also distinguished from Th2 cells by the capacity to synthesize several chemokines that included lymphotactin, monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1alpha, whereas both subsets produced macrophage inflammatory protein-1beta. Some of these chemokines as well as RANTES, MCP-3, MCP-5, and cytokine-response gene-2 (CRG-2)/IFN-inducible protein-10 (IP-10) were associated with Th1, but not Th2, pancreatic infiltrates. The data demonstrate polarization of chemokine expression by Th1 vs Th2 cells, which, within the microenvironment of the pancreas, accounts for distinctive inflammatory infiltrates that determine whether insulin-producing beta-cells are protected or destroyed.

Protection from lethal coxsackievirus-induced pancreatitis by expression of gamma interferon.

Coxsackievirus infection causes severe pancreatitis and myocarditis in humans, often leading to death in young or immunocompromised individuals. In susceptible strains of mice, coxsackievirus strain CB4 causes lethal hypoglycemia. To investigate the potential of gamma interferon (IFN-gamma) in protection and clearance of the viral infection, IFN-gamma knockout mice and transgenic (Tg) mice specifically expressing IFN-gamma in their pancreatic beta cells were infected with CB4. Lack of IFN-gamma in mice normally resistant to CB4-mediated disease resulted in hypoglycemia and rapid death. However, expression of IFN-gamma in the beta cells of Tg mice otherwise susceptible to lethal infection allowed for survival and protected them from developing the accompanying hypoglycemia. While all the mice had high levels of viral replication in their pancreata and comparable tissue pathology following viral infection, the Tg mice had significantly lower levels of virus at the peak of infection, significantly higher numbers of activated macrophages before and after infection, and less damage to their acinar tissue. Additionally, despite having increased levels of inducible nitric oxide synthetase (iNOS) expression, treatment of Tg mice with the iNOS inhibitor aminoguanidine did not alter the level of protection afforded by IFN-gamma expression. In conclusion, IFN-gamma protects from lethal coxsackievirus infection by activating macrophages in an iNOS-independent manner.

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.

Diabetes induced by Coxsackie virus: initiation by bystander damage and not molecular mimicry.

Viral induction of autoimmunity is thought to occur by either bystander T-cell activation or molecular mimicry. Coxsackie B4 virus is strongly associated with the development of insulin-dependent diabetes mellitus in humans and shares sequence similarity with the islet autoantigen glutamic acid decarboxylase. We infected different strains of mice with Coxsackie B4 virus to discriminate between the two possible induction mechanisms, and found that mice with susceptible MHC alleles had no viral acceleration of diabetes, but mice with a T cell receptor transgene specific for a different islet autoantigen rapidly developed diabetes. These results show that diabetes induced by Coxsackie virus infection is a direct result of local infection leading to inflammation, tissue damage, and the release of sequestered islet antigen resulting in the re-stimulation of resting autoreactive T cells, further indicating that the islet antigen sensitization is an indirect consequence of the viral infection.

TGF-beta1 alters APC preference, polarizing islet antigen responses toward a Th2 phenotype.

TGF-beta1, expressed in the pancreatic islets, protects the nonobese diabetic (NOD) mouse from insulin-dependent diabetes mellitus (IDDM). The islet antigen-specific T cell response of ins-TGF-beta1 mice relied on different antigen-presenting cells (APC) from those used by NOD T cells. T cells from NOD mice utilized B cells to present islet antigen, whereas T cells from ins-TGF-beta1 mice utilized macrophages. In addition, the islet antigen-specific T cell repertoire of ins-TGF-beta1 mice was distinct and deviated toward an IL-4-producing Th2 phenotype. When ins-TGF-beta1 mice were treated with anti-iL-4 antibody, islet antigen-specific IFNGamma-producing Th1 cells were unleashed, and the incidence of diabetes increased to the level of NOD mice. This suggests active suppression of a diabetogenic T cell response. This study describes a novel mechanism in which expression of TGF-beta1 in the context of self-antigen shifts APC preference, deviating T cell responses to a Th2 phenotype, 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.

Mechanism underlying counterregulation of autoimmune diabetes by IL-4.

Diabetes in nonobese diabetic (NOD) mice is an autoimmune disease characterized by the destruction of the beta cells in the pancreas. We have previously reported that transgenic expression of interleukin-4 (IL-4) counterregulates the disease process, completely protecting NOD mice from insulitis and diabetes. Here we demonstrate the presence of autoreactivity but lack of pathogenicity of the IL-4-regulated lymphocytes. The importance of T cell diversity for the protective effect of IL-4 is demonstrated through breeding with transgenic BDC2.5 mice, which have an almost exclusively monoclonal T cell repertoire. Limitation of T cell diversity abrogated the protection by IL-4. We suggest that "immune deviation" in NOD-IL-4 mice is mediated by the pancreatic tissue itself, which causes activation of distinct, nonpathogenic T cell specificities.

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.

IL-4 expression by grafts from transgenic mice fails to prevent allograft rejection.

Cell-mediated tissue destruction, such as that occurring in allograft rejection, is thought to be mediated by Th1 cells and cytokines. We have recently shown that transgenic expression of the Th2 cytokine IL-4 by pancreatic beta cells completely protects nonobese diabetic (NOD) mice from autoimmune diabetes by inducing functional tolerance among autoreactive T cells. To investigate whether local IL-4 production could also induce functional tolerance among alloreactive T cells and thus prevent allograft rejection, we transplanted pancreata from transgenic neonatal mice and their nontransgenic littermates into allogeneic hosts. Within 2 wk, recipient mice had rejected their grafts regardless of the transgene's presence or absence. Considering that the vigorous immune response induced might have prevented any effect by IL-4, we injected recipient mice with anti-CD4 and anti-CD8 mAbs, thereby depleting them of T cells and thus providing the islets with an opportunity to mature and grow. This approach indeed delayed rejection of neonatal pancreata from nontransgenic mice by >1 wk. By that time, however, pancreata from transgenic mice had also been rejected. Our results indicate that the allograft rejection response under these conditions, in contrast to the autoimmune response in NOD mice, cannot be regulated by local IL-4 production, regardless of the cytokine's impact on Th1 cells.