Constitutive differences in antioxidant defense status distinguish alloxan-resistant and alloxan-susceptible mice.

Alloxan (AL), a potent generator of superoxide and hydroxyl radicals, selectively destroys rodent pancreatic beta-cells. Alloxan-susceptible (ALS/Lt) and AL-resistant (ALR/Lt) are inbred mouse strains derived in Japan by inbreeding CD-1 (ICR) mice with concomitant selection for high or low sensitivity to a relatively low AL dose. The present study was undertaken to examine whether resistance was mediated by differences in either systemic or beta-cell antioxidant defense status. Superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPX) activities were determined in tissues of AL-untreated ALR/Lt and ALS/Lt male mice at 7 weeks of age. Specific activities of pancreatic SOD1, GR, and GPX were significantly increased in ALR/Lt mice compared with ALS/Lt mice. ALR/Lt mice further exhibited higher levels of glutathione in plasma, blood, pancreas, and liver combined with lower constitutive lipid peroxides in serum, liver, and pancreas. These results support the hypothesis that the selection process leading to the development of an AL-resistant mouse strain entailed accumulation of a gene or genes contributing to upregulated antioxidant status.     

Integrin-linked kinase regulates inducible nitric oxide synthase and cyclooxygenase-2 expression in an NF-kappa B-dependent manner.

Nitric oxide (NO) and prostaglandins are produced as a result of the stimulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2, respectively, in response to cytokines or lipopolysaccharide (LPS). We demonstrate that the activity of integrin-linked kinase (ILK) is stimulated by LPS activation in J774 macrophages. Inhibition of ILK activity by dominant-negative ILK or a highly selective small molecule ILK inhibitor, in epithelial cells or LPS-stimulated J774 cells and murine macrophages, resulted in inhibition of iNOS expression and NO synthesis. LPS stimulates the phosphorylation of IkappaB on Ser-32 and promotes its degradation. Inhibition of ILK suppressed this LPS-stimulated IkappaB phosphorylation and degradation. Similarly, ILK inhibition suppressed the LPS-stimulated iNOS promoter activity. Mutation of the NF-kappaB sites in the iNOS promoter abolished LPS- and ILK-mediated regulation of iNOS promoter activity. Overexpression of ILK-stimulated NF-kappaB activity and inhibition of ILK or protein kinase B (PKB/Akt) suppressed this activation. We conclude that ILK can regulate NO production in macrophages by regulating iNOS expression through a pathway involving PKB/Akt and NF-kappaB. Furthermore, we also demonstrate that ILK activity is required for LPS stimulated cyclooxygenase-2 expression in murine and human macrophages. These findings implicate ILK as a potential target for anti-inflammatory applications.     

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.     

NF-kappa B hyperactivation has differential effects on the APC function of nonobese diabetic mouse macrophages

Type 1 diabetes is characterized by a chronic inflammatory response resulting in the selective destruction of the insulin-producing beta cells. We have previously demonstrated that dendritic cells (DCs) prepared from nonobese diabetic (NOD) mice, a model for spontaneous type 1 diabetes, exhibit hyperactivation of NF-kappaB resulting in an increased capacity to secrete proinflammatory cytokines and stimulate T cells compared with DCs of nondiabetic strains of mice. In the current study, the activational status of NF-kappaB and its role in regulating the APC function of macrophages (Mphi) prepared from NOD, nonobese resistant (NOR), and BALB/c mice was investigated. Independent of the stimulus, splenic and bone marrow-derived Mphi prepared from NOD mice exhibited increased NF-kappaB activation relative to NOR and BALB/c Mphi. This hyperactivation was detected for different NF-kappaB complexes and correlated with increased IkappaBalpha degradation. Furthermore, increased NF-kappaB activation resulted in an enhanced capacity of NOD vs NOR or BALB/c Mphi to secrete IL-12(p70), TNF-alpha, and IL-1alpha, which was inhibited upon infection with an adenoviral recombinant encoding a modified form of IkappaBalpha. In contrast, elevated NF-kappaB activation had no significant effect on the capacity of NOD Mphi to stimulate CD4(+) or CD8(+) T cells in an Ag-specific manner. These results demonstrate that in addition to NOD DCs, NOD Mphi exhibit hyperactivation of NF-kappaB, which correlates with an increased ability to mediate a proinflammatory response. Furthermore, NF-kappaB influences Mphi APC function by regulating cytokine secretion but not T cell stimulation.     

Epidermal growth factor and interleukin-1beta synergistically stimulate the production of nitric oxide in rat intestinal epithelial cells

Epidermal growth factor (EGF) is one of the trophic factors for intestinal adaptation after small bowel transplantation (SBT). A recent report indicates that nitric oxide (NO) has cytoprotective effects on bacterial translocation (BT) after SBT. We hypothesized that EGF stimulates the expression of the inducible NO synthase (iNOS) gene in the graft after SBT, followed by increased production of NO, resulting in the decrease of BT. Intestinal epithelial cells (IEC)-6 were treated with EGF and/or IL-1beta in the presence and absence of phosphatidylinositol 3-kinase (PI3-kinase) and EGF receptor kinase inhibitors (LY-294002 and tyrphostin A25). The induction of NO production and iNOS and its signal molecules, including the inhibitory protein of NF-kappaB (IkappaB), NF-kappaB, and Akt, were analyzed. IL-1beta stimulated the degradation of IkappaB and the activation of NF-kappaB but had no effect on iNOS induction. EGF, which had no effect on the NF-kappaB activation and iNOS induction, stimulated the upregulation of type 1 IL-1 receptor (IL-1R1) through PI3-kinase/Akt. Simultaneous addition of EGF and IL-1beta stimulated synergistically the induction of iNOS, leading to the increased production of NO. Our results indicate that EGF and IL-1beta stimulate two essential signals for iNOS induction in IEC-6 cells: the upregulation of IL-1R1 through PI3-kinase/Akt and the activation of NF-kappaB through IkappaB kinase, respectively. Simultaneous addition of EGF and IL-1beta can enhance the production of NO, which may contribute to the cytoprotective effect of EGF against intestinal injury.     

Commonalities of genetic resistance to spontaneous autoimmune and free radical--mediated diabetes

ALR/Lt, a NOD-related mouse strain, was selected for resistance to alloxan free radical-mediated diabetes (ALD). Despite extensive genomic identity with NOD (>70%), ALR mice display strong resistance to autoimmune type 1 diabetes (T1D) due to both an unusual elevation in systemic antioxidant defenses and a reduction in cellular ROS production that extends to the beta cell level. Reciprocal backcross to NOD previously linked the ALR-derived T1D resistance to Chr. 3, 8, and 17 as well as to the ALR mt-Nd2(a) allele encoded by the mitochondrial genome (mtDNA). To determine whether any of the ALR-derived loci protecting against T1D also protected against ALD, 296 six-week-old F2 mice from reciprocal outcrosses were alloxan-treated and assessed for diabetes onset, and a genome-wide scan (GWS) was conducted. GWS linked mt-Nd2 as well as three nuclear loci with alloxan-induced diabetes. A dominant ALR-derived ALD resistance locus on Chr. 8 colocalized with the ALR-derived T1D resistance locus identified in the previous backcross analysis. In contrast, whereas ALR contributed a novel T1D resistance locus on Chr. 3 marked by Susp, a more proximal ALR-derived region marked by Il-2 contributed ALD susceptibility, not resistance. In addition, a locus was mapped on Chr. 2, where heterozygosity provided heightened susceptibility. Tests for alloxan sensitivity in ALR conplastic mice encoding the NOD mt-Nd2(c) allele and NOD mice congenic for the protective Chr. 8 locus supported our mapping results. Alloxan sensitivity was increased in ALR.mt(NOD) mice, whereas it was decreased by congenic introduction of ALR genome on Chr. 8 into NOD. These data demonstrate both similarities and differences in the genetic control of T1D versus ROS-induced diabetes.     

A pyrrolidinone derivative inhibits cytokine-induced iNOS expression and NF-kappaB activation by preventing phosphorylation and degradation of IkappaB-alpha

We previously showed that 1-[3-(3-pyridyl)-acryloyl]-2-pyrrolidinone hydrochloride (N2733) inhibits lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF)-alpha secretion and improves the survival of endotoxemic mice. Since overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) in vascular smooth muscle cells (VSMCs) is largely responsible for the development of endotoxemic shock, and iNOS gene expression is mainly regulated by LPS and inflammatory cytokines, we studied whether or not N2733 affects interleukin (IL)-1beta-induced iNOS gene expression, NF-kappaB activation, and NF-kappaB inhibitor (IkappaB)-alpha degradation in cultured rat VSMCs. N2733 dose-dependently (10-100 microM) inhibited IL-1beta-stimulated NO production, and decreased IL-1beta-induced iNOS mRNA and protein expression, as found on Northern and Western blot analyses, respectively. Gel shift assay and an immunocytochemical study showed that N2733 inhibited IL-1beta-induced NF-kappaB activation and its nuclear translocation. Western blot analyses involving anti-IkappaB-alpha and anti-phospho IkappaB-alpha antibodies showed that IL-1beta induced transient degradation of IkappaB-alpha preceded by the rapid appearance of phosphorylated IkappaB-alpha, both of which were markedly blocked by N2733. N2733 blocked IL-1beta-induced phosphorylated IkappaB-alpha even in the presence of a proteasome inhibitor (MG115). Immunoblot analysis involving anti-IkappaB kinase (IKK)-alpha and anti-phosphoserine antibodies revealed that N2733 inhibited IL-1beta-induced IKK-alpha phosphorylation, whereas N2733 had no inhibitory effect on IL-1beta-stimulated p42/p44 MAP kinase or p38 MAP kinase activity. Our results suggest that the inhibitory action of N2733 toward IL-1beta-induced NF-kappaB activation and iNOS expression is due to its blockade of the upstream signal(s) leading to IKK-alpha activation, and subsequent phosphorylation and degradation of IkappaB-alpha in rat VSMCs.     

Immunoregulation during disease progression in prediabetic NOD mice: inverse expression of arginase and prostaglandin H synthase 2 vs. interleukin-15.

Non-obese diabetic (NOD) mice spontaneously develop insulin dependent diabetes due to autoimmune destruction of beta-cells. The progression of insulitis can be accelerated and synchronized in the pancreas by a single injection of 250 mg/kg cyclophosphamide. In this study, we will report on three immune mediators that were not known to be expressed during insulitis until now. Early insulitis in ten-week-old female NOD mice was associated with strong expression of prostaglandin H synthase 2 in the pancreas and of arginase, an antagonist enzyme of the inducible NO synthase. After acceleration of insulitis progression by cyclophosphamide, expression of the two enzymes was downregulated within 24 h. There was strong concomitant upregulation of IL-15 gene expression that preceded lymphocyte invasion of islets and a rise of IFN-gamma mRNA levels by several days. The comparison of individual pancreata showed that the expression of IL-12 and IL-18 mRNA closely correlated with levels of IL-15 gene expression. We conclude that arginase and prostaglandin H synthase 2 expression is associated with peri-insulitis, while IL-15 is a candidate cytokine in driving destructive insulitis, as it elicits Th1-cytotoxic responses in lymphoid as well as in non-lymphoid immune cells and is unusually resistant to downregulation by antagonistic cytokines. This is the first report on arginase, prostaglandin H synthase 2 and IL-15 expression in pancreatic lesions of prediabetic NOD mice.     

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

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

Genetic analysis of resistance to Type-1 Diabetes in ALR/Lt mice,a NOD-related strain with defenses against autoimmune-mediated diabetogenic stress

ALR mice are closely related to type-1 diabetes mellitus (T1DM)-prone NOD mice. The ALR genome confers systemically elevated free radical defenses, dominantly protecting their pancreatic islets from free radical generating toxins, cytotoxic cytokines, and diabetogenic T cells. The ALR major histocompatibility complex (MHC) (H2^gx haplotype) is largely, but not completely identical with the NOD H2^g7 haplotype, sharing alleles from H2-K through the class II and distally into the class III region. This same H2^gx haplotype in the related CTS strain was linked to the Idd16 resistance locus. In the present study, ALR was outcrossed to NOD to fine map the Idd16 locus and establish chromosomal regions carrying other ALR non-MHC-linked resistance loci. To this end, 120 (NOD×ALR)×NOD backcross progeny females were monitored for T1DM and genetic linkage analysis was performed on all progeny using 88 markers covering all chromosomes. Glucosuria or end-stage insulitis developed in 32 females, while 88 remained both aglucosuria and insulitis free. Three ALR-derived resistance loci segregated. As expected, one mapped to Chromosome 17, with peak linkage mapping just proximal to H2-K. A novel resistance locus mapped to Chr 8. A pairwise scan for interactions detected a significant interaction between the loci on Chr 8 and Chr 17. On Chr 3, resistance segregated with a marker between previously described Idd loci and coinciding with an independently mapped locus conferring a suppressed superoxide burst by ALR neutrophils (Susp). These results indicate that the Idd16 resistance allele, defined originally by linkage to the H2^gx haplotype of CTS, is immediately proximal to H2-K. Two additional ALR-contributed resistance loci may be ALR-specific and contribute to this strain's ability to dissipate free-radical stress.     

UV as an amplifier rather than inducer of NF-kappaB activity

Inflammatory activation of NF-kappaB involves the stimulus-induced degradation of the NF-kappaB-bound inhibitor IkappaB via the IkappaB kinase (IKK). In response to UV irradiation, however, the mechanism and function of NF-kappaB activation remain unclear. Using a combined biochemical, genetic, and computational modeling approach, we delineate a dual requirement for constitutive IKK-dependent and IKK-independent IkappaB degradation pathways in conjunction with UV-induced translational inhibition. Interestingly, we find that the high homeostatic turnover of IkappaB in resting cells renders the NF-kappaB system remarkably resistant to metabolic stresses, but the two degradation pathways critically and differentially tune NF-kappaB responsiveness to UV. Indeed, in the context of low chronic inflammation that accelerates NF-kappaB-bound IkappaB degradation, UV irradiation results in dramatic NF-kappaB activation. Our work suggests that the human health relevance of NF-kappaB activation by UV lies with cellular homeostatic states that are associated with pathology rather than with healthy physiology.     

Role of inflammatory infiltrate in activation and effector function of cloned islet reactive nonobese diabetic CD8+ T cells: involvement of a nitric oxide-dependent pathway

To investigate how CD8+ T cells interact with beta cells and local inflammatory cells in islets, we have isolated CD8+ T cell clones from nonobese diabetic (NOD) spleen that recognize and destroy both islets and the NOD insulinoma cell line NIT-1. The clones destroyed NOD islets with pre-existing inflammation better than islets without signs of inflammation. Islets from NOD-scid mice were destroyed only poorly, but that could be improved by adding IL-7 to the assay. Anti-IFN-gamma Abs inhibited destruction of infiltrated islets. Single islets were effective stimulators of IFN-gamma production by cloned CD8+ T cells, which varied >50-fold depending on the degree of islet infiltration. This effect of the islet mononuclear infiltrate could be mimicked by adding spleen cells to NIT-1 cells, which augmented IFN-gamma production above the level stimulated by NIT-1 cells alone. The enhancing effect of spleen cells could be attributed to their macrophage subpopulation and was not MHC restricted, although recognition of islet Ag by cloned CD8+ T cells and subsequent islet destruction was restricted to islets expressing H-2Db molecules. An inhibitor of inducible NO synthase inhibited destruction of inflamed islets by cloned CD8+ T cells. We propose that macrophages in inflamed islets provide a form of bystander costimulation of beta cell-specific CD8+ T cells. CD8+ T cells respond to Ag and costimulation by producing IFN-gamma that activates macrophages. Activated macrophages facilitate islet destruction by CD8+ T cells through a NO synthesis-dependent pathway.     

Genetic control of neutrophil superoxide production in diabetes-resistant ALR/Lt mice

The neutrophil oxidative burst reaction differentiates ALR/Lt mice, known for an unusual systemic elevation of antioxidant defenses, from ALS/Lt mice, a related strain known for reduced ability to withstand oxidative stress. Neutrophils from marrow of ALS mice produced a normal neutrophil oxidative burst following phorbol ester stimulation. In contrast, ALR mice exhibited a markedly suppressed superoxide burst. F1 progeny from reciprocal outcrosses between ALR and ALS mice exhibited an intermediate burst level, higher than ALR but significantly lower than ALS. To elucidate the genetic basis for this strain difference, F1 mice were backcrossed to ALS mice, and marrow neutrophils isolated from the progeny were phenotyped for oxidative burst capacity. A genome-wide sweep using polymorphic markers distinguishing the two parental strains was performed to map the trait. A 1:1 phenotypic distribution was observed, and a locus (Suppressor of superoxide production, Susp) controlling this phenotype was mapped to Chromosome 3 near D3Mit241 at 33.1 cM. This locus probably represents an important regulatory element in the overall ALR strain resistance to oxidative stress, since diminished ability to mount a neutrophil burst in backcross segregants correlated with elevated hepatic superoxide dismutase 1 (SOD1) activity, an ALR strain characteristic.     

Transgenic expression of dominant-negative Fas-associated death domain protein in beta cells protects against Fas ligand-induced apoptosis and reduces spontaneous diabetes in nonobese diabetic mice

In type 1 diabetes, many effector mechanisms damage the beta cell, a key one being perforin/granzyme B production by CD8(+) T cells. The death receptor pathway has also been implicated in beta cell death, and we have therefore generated NOD mice that express a dominant-negative form of the Fas-associated death domain protein (FADD) adaptor to block death receptor signaling in beta cells. Islets developed normally in these animals, indicating that FADD is not necessary for beta cell development as it is for vasculogenesis. beta cells from the transgenic mice were resistant to killing via the Fas pathway in vitro. In vivo, a reduced incidence of diabetes was found in mice with higher levels of dominant-negative FADD expression. This molecule also blocked signals from the IL-1R in culture, protecting isolated islets from the toxic effects of cytokines and also marginally reducing the levels of Fas up-regulation. These data support a role for death receptors in beta cell destruction in NOD mice, but blocking the perforin/granzyme pathway would also be necessary for dominant-negative FADD to have a beneficial clinical effect.