Shane T. Grey

Expression of heme oxygenase-1 can determine cardiac xenograft survival

The rejection of concordant xenografts, such as mouse-to-rat cardiac xenografts, is very similar to the delayed rejection of porcine-to-primate discordant xenografts. In concordant models, this type of rejection is prevented by brief complement inhibition by cobra venom factor (CVF) and sustained T-cell immunosuppression by cyclosporin A (CyA) (refs. 7, 8, 9, 10). Mouse hearts that survive indefinitely in rats treated with CVF plus CyA express the anti-inflammatory gene heme oxygenase-1 (HO-1) in their endothelial cells and smooth muscle cells. The anti-inflammatory properties of HO-1 are thought to rely on the ability of this enzyme to degrade heme and generate bilirubin, free iron and carbon monoxide. Bilirubin is a potent anti-oxidant, free iron upregulates the transcription of the cytoprotective gene, ferritin, and carbon monoxide is thought to be essential in regulating vascular relaxation in a manner similar to nitric oxide. We show here that the expression of the HO-1 gene is functionally associated with xenograft survival, and that rapid expression of HO-1 in cardiac xenografts can be essential to ensure long-term xenograft survival.

In vivo expansion of T reg cells with IL-2–mAb complexes: induction of resistance to EAE and long-term acceptance of islet allografts without immunosuppression

Via a transcription factor, Foxp3, immunoregulatory CD4+CD25+ T cells (T reg cells) play an important role in suppressing the function of other T cells. Adoptively transferring high numbers of T reg cells can reduce the intensity of the immune response, thereby providing an attractive prospect for inducing tolerance. Extending our previous findings, we describe an in vivo approach for inducing rapid expansion of T reg cells by injecting mice with interleukin (IL)-2 mixed with a particular IL-2 monoclonal antibody (mAb). Injection of these IL-2–IL-2 mAb complexes for a short period of 3 d induces a marked (>10-fold) increase in T reg cell numbers in many organs, including the liver and gut as well as the spleen and lymph nodes, and a modest increase in the thymus. The expanded T reg cells survive for 1–2 wk and are highly activated and display superior suppressive function. Pretreating with the IL-2–IL-2 mAb complexes renders the mice resistant to induction of experimental autoimmune encephalomyelitis; combined with rapamycin, the complexes can also be used to treat ongoing disease. In addition, pretreating mice with the complexes induces tolerance to fully major histocompatibility complex–incompatible pancreatic islets in the absence of immunosuppression. Tolerance is robust and the majority of grafts are accepted indefinitely. The approach described for T reg cell expansion has clinical potential for treating autoimmune disease and promoting organ transplantation.

Superoxide-mediated activation of uncoupling protein 2 causes pancreatic β cell dysfunction

Failure to secrete adequate amounts of insulin in response to increasing concentrations of glucose is an important feature of type 2 diabetes. The mechanism for loss of glucose responsiveness is unknown. Uncoupling protein 2 (UCP2), by virtue of its mitochondrial proton leak activity and consequent negative effect on ATP production, impairs glucose-stimulated insulin secretion. Of interest, it has recently been shown that superoxide, when added to isolated mitochondria, activates UCP2-mediated proton leak. Since obesity and chronic hyperglycemia increase mitochondrial superoxide production, as well as UCP2 expression in pancreatic beta cells, a superoxide-UCP2 pathway could contribute importantly to obesity- and hyperglycemia-induced beta cell dysfunction. This study demonstrates that endogenously produced mitochondrial superoxide activates UCP2-mediated proton leak, thus lowering ATP levels and impairing glucose-stimulated insulin secretion. Furthermore, hyperglycemia- and obesity-induced loss of glucose responsiveness is prevented by reduction of mitochondrial superoxide production or gene knockout of UCP2. Importantly, reduction of superoxide has no beneficial effect in the absence of UCP2, and superoxide levels are increased further in the absence of UCP2, demonstrating that the adverse effects of superoxide on beta cell glucose sensing are caused by activation of UCP2. Therefore, superoxide-mediated activation of UCP2 could play an important role in the pathogenesis of beta cell dysfunction and type 2 diabetes.

BAFF and MyD88 signals promote a lupuslike disease independent of T cells

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the production of autoantibodies. However, the underlying cause of disease appears to relate to defects in T cell tolerance or T cell help to B cells. Transgenic (Tg) mice overexpressing the cytokine B cell–activating factor of the tumor necrosis factor family (BAFF) develop an autoimmune disorder similar to SLE and show impaired B cell tolerance and altered T cell differentiation. We generated BAFF Tg mice that were completely deficient in T cells, and, surprisingly, these mice developed an SLE-like disease indistinguishable from that of BAFF Tg mice. Autoimmunity in BAFF Tg mice did, however, require B cell–intrinsic signals through the Toll-like receptor (TLR)–associated signaling adaptor MyD88, which controlled the production of proinflammatory autoantibody isotypes. TLR7/9 activation strongly up-regulated expression of transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), which is a receptor for BAFF involved in B cell responses to T cell–independent antigens. Moreover, BAFF enhanced TLR7/9 expression on B cells and TLR-mediated production of autoantibodies. Therefore, autoimmunity in BAFF Tg mice results from altered B cell tolerance, but requires TLR signaling and is independent of T cell help. It is possible that SLE patients with elevated levels of BAFF show a similar basis for disease.

Role for Activating Transcription Factor 3 in Stress-Induced β-Cell Apoptosis

ABSTRACT Activating transcription factor 3 (ATF3) is a stress-inducible gene and encodes a member of the ATF/CREB family of transcription factors. However, the physiological significance of ATF3 induction by stress signals is not clear. In this report, we describe several lines of evidence supporting a role of ATF3 in stress-induced β-cell apoptosis. First, ATF3 is induced in β cells by signals relevant to β-cell destruction: proinflammatory cytokines, nitric oxide, and high concentrations of glucose and palmitate. Second, induction of ATF3 is mediated in part by the NF-κB and Jun N-terminal kinase/stress-activated protein kinase signaling pathways, two stress-induced pathways implicated in both type 1 and type 2 diabetes. Third, transgenic mice expressing ATF3 in β cells develop abnormal islets and defects secondary to β-cell deficiency. Fourth, ATF3 knockout islets are partially protected from cytokine- or nitric oxide-induced apoptosis. Fifth, ATF3 is expressed in the islets of patients with type 1 or type 2 diabetes, and in the islets of nonobese diabetic mice that have developed insulitis or diabetes. Taken together, our results suggest ATF3 to be a novel regulator of stress-induced β-cell apoptosis.

The BAFF/APRIL System: An Important Player in Systemic Rheumatic Diseases

Many rheumatic diseases have an autoimmune basis, characterized by organ-specific inflammation and tissue destruction. Diseases such as rheumatoid arthritis, systemic lupus erythematosus or Sjögrens syndrome often associate with abnormal B cell function and the production of various autoantibodies. B cell activating factor belonging to the TNF family (BAFF) is a B cell survival factor essential for B cell maturation, but also contributes to autoimmunity when overexpressed in mice. In addition, elevated levels of BAFF have been detected in the serum of patients with various rheumatic diseases, suggesting a role for this factor in these pathologies. BAFF has additional functions that may be important in rheumatic diseases. For instance, excess BAFF leads to the expansion of a subset of B cells named marginal zone (MZ) B cells, a cell type able to activate naïve T cells. In addition, expansion of the MZ B cell population correlates with certain autoimmune diseases, and these cells have been detected in inflamed tissues in mice and humans. Recently, BAFF was shown to also stimulate T cell activation, an aspect that may also contribute to autoimmunity. Finally, BAFF has emerged as a potent survival factor for B cell lymphomas and as such may be involved in promoting B cell cancers. This result possibly offers an explanation for the occasional lymphoma complication observed in a subset of patients with certain rheumatic diseases, particularly Sjögrens syndrome. New elements about BAFF biology indicate that this factor may be involved in a wider range of pathologies than first anticipated, and inhibitors of this factor are likely to provide attractive new treatments for rheumatic diseases and B cell lymphomas.

Hypoxia-inducible factor-1α regulates β cell function in mouse and human islets

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor that regulates cellular stress responses. While the levels of HIF-1alpha protein are tightly regulated, recent studies suggest that it can be active under normoxic conditions. We hypothesized that HIF-1alpha is required for normal beta cell function and reserve and that dysregulation may contribute to the pathogenesis of type 2 diabetes (T2D). Here we show that HIF-1alpha protein is present at low levels in mouse and human normoxic beta cells and islets. Decreased levels of HIF-1alpha impaired glucose-stimulated ATP generation and beta cell function. C57BL/6 mice with beta cell-specific Hif1a disruption (referred to herein as beta-Hif1a-null mice) exhibited glucose intolerance, beta cell dysfunction, and developed severe glucose intolerance on a high-fat diet. Increasing HIF-1alpha levels by inhibiting its degradation through iron chelation markedly improved insulin secretion and glucose tolerance in control mice fed a high-fat diet but not in beta-Hif1a-null mice. Increasing HIF-1alpha levels markedly increased expression of ARNT and other genes in human T2D islets and improved their function. Further analysis indicated that HIF-1alpha was bound to the Arnt promoter in a mouse beta cell line, suggesting direct regulation. Taken together, these findings suggest an important role for HIF-1alpha in beta cell reserve and regulation of ARNT expression and demonstrate that HIF-1alpha is a potential therapeutic target for the beta cell dysfunction of T2D.

BAFF Augments Certain Th1-Associated Inflammatory Responses

B cell-activating factor belonging to the TNF family (BAFF; BLyS) is a critical regulator of B cell maturation and survival, and its overexpression in BAFF transgenic (Tg) mice results in the development of autoimmune disorders. BAFF also affects T cell function through binding to one of the BAFF receptors, BAFF-R. Using BAFF Tg mice, we examined a typical Th1-mediated response, the cutaneous delayed-type hypersensitivity reaction, and found a much greater degree of paw swelling and inflammation than in control mice. Importantly, delayed-type hypersensitivity scores correlated directly with BAFF levels in serum. Conversely, in a Th2-mediated model of allergic airway inflammation, BAFF Tg mice were largely protected and showed markedly reduced Ag-specific T cell proliferation and eosinophil infiltration associated with the airways. Thus, local and/or systemically distributed BAFF affects Th1 and Th2 responses and impacts on the course of some T cell-mediated inflammatory reactions. Our results are consistent with the idea that BAFF augments T cell as well as B cell responses, particularly Th1-type responses. Results in BAFF Tg mice may reflect the situation in certain autoimmune patients or virally infected individuals, because BAFF levels in blood are comparable.

Binding of activated protein c to a specific receptor on human mononuclear phagocytes inhibits intracellular calcium signaling and monocyte-dependent proliferative responses

Upon activation, mononuclear phagocytes (Mphi) play key roles in the development of septic shock and multiple host immune responses, but details of the regulation of Mphi activation are little understood. We recently showed that the physiologic anticoagulant molecule, activated protein C (APC), blocks responses of human blood Mphi, alveolar Mphi, or THP-1 cells induced by LPS, IFN-gamma, or PMA, including TNF-alpha production and down-regulation of several LPS binding-related proteins. We now report a possible mechanism of action through inhibition of the rapid intracellular calcium signaling that occurs at the onset of Mphi activation, and characterization of a specific Mphi receptor for APC. Flow cytometry studies using Fluo-3 showed that Mph activation by Fc-receptor cross-linking or rIFN-gamma caused a rapid increase in free intracellular calcium, a primary event in multiple signal transduction pathways, which was blocked by pretreatment with APC. Consistent with this, addition of APC inhibited PHA-induced T cell proliferation in a dose- and time-dependent manner. Peak suppression (> 70%) required addition of APC within the first hour of 72 hr cocultures of Mphi and lymphocytes, and proliferative responses were not restored by addition of IL-2 or TNF-alpha. Biochemical studies showed that 125I-labeled APC bound specifically to M phi in a time-dependent and saturable manner. Scatchard analysis indicated there were 180,690 binding sites for APC per cell, which were of high affinity (Kd value of 12.9 mM). Binding of 125I-APC was doubled by activation of Mphi with LPS, and bound APC was not displaced by the zymogen, protein C (PC), or by enzymatically inactive (diisopropyl fluorophosphate- or PPACK-treated) APC, indicating an absolute requirement for the active site of APC in its binding to Mphi. APC binding was blocked by a polyclonal Ab to human PC/APC, but not by protein S, factor Va or Xa, or a polyclonal antithrombomodulin antibody. When 125I-APC was crosslinked to its receptor, immunoprecipitated and analyzed by SDS-PAGE under reducing conditions, a covalent complex (110-115 kD) of 125I-APC (62 kD) and its receptor was seen. In addition, a Mphi membrane protein of 50-55 kD, as determined by SDS-PAGE, was affinity-purified using an APC-Affigel column, and confirmed by ligand binding. Taken together, our findings document the presence of a M phi surface receptor for APC, which appears distinct from a recently described endothelial receptor for PC and APC, and which may be involved in the inhibitory effects of APC on activation of human Mphi, including Mphi-dependent T cell proliferation.

Marginal-Zone B-Cells of Nonobese Diabetic Mice Expand With Diabetes Onset, Invade the Pancreatic Lymph Nodes, and Present Autoantigen to Diabetogenic T-Cells

OBJECTIVE—B-cells are important for disease pathogenesis in the nonobese diabetic (NOD) mouse model of type 1 diabetes. Recent studies demonstrate that marginal-zone B-cells (MZBs), which connect innate with adaptive immune responses, are increased in NOD mice. However, beyond this, the contribution of different B-cell subsets to diabetes pathogenesis is poorly understood. RESEARCH DESIGN AND METHODS—To better understand the role of different B-cell subsets in the etiology of type 1 diabetes, we have examined the MZB compartment in NOD mice, with respect to their number, distribution, and function. RESULTS—We demonstrate that splenic MZB numbers in female NOD mice undergo a marked, approximately threefold expansion between ∼12 and 16 weeks of age, coincident with the onset of frank diabetes. Functionally, NOD MZBs are hyperresponsive to toll-like receptor 9 ligation and CD40 ligation, as well as sphingosine-1-phosphate–dependent chemotactic cues, suggesting an increased sensitivity to selective innate- and activation-induced stimuli. Intriguingly, at 16 weeks of age, ∼80% of female NOD mice present with MZB-like cells in the pancreatic lymph node (PLN). These MZB-like cells express major histocompatibility complex class II and high levels of CD80 and CD86, and their presence in the PLN is associated with an increased frequency of activated Vβ4+ CD4+ T-cells. Significantly, we demonstrate that purified MZBs are able to present the autoantigen insulin to diabetogenic T-cells. CONCLUSIONS—These data are consistent with MZBs contributing to the pathogenesis of type 1 diabetes as antigen-presenting cells. By integrating innate-derived inflammatory signals with the activation of autoreactive T-cells, MZBs may help to direct T-cell responses against β-cell self-constituents.