The proapoptotic BH3-only proteins Bim and Puma are downstream of endoplasmic reticulum and mitochondrial oxidative stress in pancreatic islets in response to glucotoxicity.

Apoptosis of pancreatic beta cells is a feature of type 2 diabetes and its prevention may have therapeutic benefit. High glucose concentrations induce apoptosis of islet cells, and this requires the proapoptotic Bcl-2 homology domain 3 (BH3)-only proteins Bim and Puma. We studied the stress pathways induced by glucotoxicity in beta cells that result in apoptosis. High concentrations of glucose or ribose increased expression of the transcription factor CHOP (C/EBP homologous protein) but not endoplasmic reticulum (ER) chaperones, indicating activation of proapoptotic ER stress signaling. Inhibition of ER stress prevented ribose-induced upregulation of Chop and Puma mRNA, and partially protected islets from glucotoxicity. Loss of Bim or Puma partially protected islets from the canonical ER stressor thapsigargin. The antioxidant N-acetyl-cysteine also partially protected islets from glucotoxicity. Islets deficient in both Bim and Puma, but not Bim or Puma alone, were significantly protected from killing induced by the mitochondrial reactive oxygen species donor rotenone. Our data demonstrate that high concentrations of glucose induce ER and oxidative stress, which causes cell death mediated by Bim and Puma. We observed significantly higher Bim and Puma mRNA in islets of human donors with type 2 diabetes. This indicates that inhibition of Bim and Puma, or their inducers, may prevent beta-cell destruction in type 2 diabetes.

Discovery of molecular pathways mediating 1,25-dihydroxyvitamin D3 protection against cytokine-induced inflammation and damage of human and male mouse islets of Langerhans.

Protection against insulitis and diabetes by active vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in nonobese diabetic mice has until now mainly been attributed to its immunomodulatory effects, but also protective effects of this hormone on inflammation-induced ß-cell death have been reported. The aim of this study was to clarify the molecular mechanisms by which 1,25(OH)2D3 contributes to ß-cell protection against cytokine-induced ß-cell dysfunction and death. Human and mouse islets were exposed to IL-1ß and interferon-γ in the presence or absence of 1,25(OH)2D3. Effects on insulin secretion and ß-cell survival were analyzed by glucose-stimulated insulin release and electron microscopy or Hoechst/propidium iodide staining, respectively. Gene expression profiles were assessed by Affymetrix microarrays. Nuclear factor-κB activity was tested, whereas effects on secreted chemokines/cytokines were confirmed by ELISA and migration studies. Cytokine exposure caused a significant increase in ß-cell apoptosis, which was almost completely prevented by 1,25(OH)2D3. In addition, 1,25(OH)2D3 restored insulin secretion from cytokine-exposed islets. Microarray analysis of murine islets revealed that the expression of approximately 4000 genes was affected by cytokines after 6 and 24 hours (n = 4; >1.3-fold; P < .02), of which nearly 250 genes were modified by 1,25(OH)2D3. These genes belong to functional groups involved in immune response, chemotaxis, cell death, and pancreatic ß-cell function/phenotype. In conclusion, these findings demonstrate a direct protective effect of 1,25(OH)2D3 against inflammation-induced ß-cell dysfunction and death in human and murine islets, with, in particular, alterations in chemokine production by the islets. These effects may contribute to the beneficial effects of 1,25(OH)2D3 against the induction of autoimmune diabetes.

A proteomic approach on the effects of TX527, a 1α,25-dihydroxyvitamin D3 analog, in human T lymphocytes.

1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3), and its analogs (i.e. 14,20-bis-epi-19-nor-23-yne-1α,25(OH)2D3 - TX527) have been shown to prevent autoimmunity and prolong islet graft survival in the non-obese diabetic (NOD) mouse. Their effects are mediated by their action on various immune cell types, such as dendritic cells (DC) and T cells. We have previously reported important direct effects of TX527 on human T cells, on their cytokine/chemokine profiles, T regulatory cell markers, homing characteristics and chemotaxis. In order to fully understand the molecular mechanisms underlying the beneficial properties of TX527 on human T cells, we applied here 2-dimensional difference gel electrophoresis (2-D DIGE) to analyze the global protein alterations induced by TX527 on human synchronized T cells. We detected differential expression of 64 protein spots upon TX527 treatment, of which 65.6% could be successfully identified using tandem mass spectrometry (MALDI-TOF/TOF). The identified proteins function in various processes, such as metabolism and energy pathways, cytoskeleton and protein metabolism. When comparing the proteomics data to our previously performed microarray data on the same set of cells, we found an overlap of 17 different mRNAs/proteins. For some of these (e.g. PSME2, HSPA8), the direction of regulation was not similar, hereby reinforcing the important role of post-transcriptional/translational processes in the functionality of proteins. In addition, although 2-D DIGE offers the possibility of picking up post-translational processes, it lacks the ability to detect molecules with extreme molecular weight (MW) and isoelectrical point (pI) values, or very low abundant/hydrophobic proteins. This study highlights therefore the importance of combining different experimental approaches to obtain a complete picture of the underlying mechanisms and general processes being affected in T cells upon TX527 treatment. These processes lead altogether to the generation of T cells with interesting immunomodulatory features for clinical applications in the treatment of autoimmune diseases or in the prevention of graft rejection. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Glucagon-like peptide-1: modulator of ß-cell dysfunction and death.

Glucagon-like peptide-1 (GLP-1) is one of the hormones responsible for the incretin effect, a term that refers to the observation that orally administered glucose results in a larger increase in plasma insulin levels and insulin-dependent decrease in blood glucose concentration when compared to the same amount of glucose given intravenously. GLP-1 is secreted mainly by gut endocrine L-cells and is released under the control of carbohydrates, proteins and lipids. Upon secretion, GLP-1 targets different cell types and exerts a wide variety of actions such as potentiation of glucose-stimulated insulin secretion, reduction of appetite, delay of gastric emptying and increase in ß-cell mass. These beneficial effects have resulted in the application of GLP-1-based therapies in patients with type 2 diabetes, but also exploitation of its effects in type 1 diabetes is being envisaged. In this review, we focus on the different, short- and long-term action mechanisms of GLP-1 with specific emphasis on its role as a modulator of ß-cell function and survival.

Unraveling the effects of 1,25OH2D3 on global gene expression in pancreatic islets.

INTRODUCTION: Vitamin D deficiency has been linked to type 1 and 2 diabetes, whereas supplementation may prevent both diseases. However, the extent of the effects of vitamin D or its metabolites directly on pancreatic islets is still largely unknown. The aim of the present study was to investigate how active vitamin D, 1,25(OH)2D3, affects beta cells directly by establishing its effects on global gene expression in healthy murine islets. MATERIALS AND METHODS: Pancreatic islets were isolated from 2 to 3 week old C57BL/6 mice and cultured in vitro with 1,25(OH)2D3 or vehicle for 6 and 24h. Total RNA was extracted from the islets and the effects on global gene expression were analyzed using Affymetrix microarrays. RESULTS AND DISCUSSION: Exposure to 1,25(OH)2D3 compared to vehicle resulted in 306 and 151 differentially expressed genes after 6 and 24h, respectively (n=4, >1.3-fold, p<0.02). Of these 220 were up-regulated, whereas 86 displayed a decreased expression after 6h. Furthermore, expression levels were increased for 124 and decreased for 27 genes following 24h of exposure. Formation of intercellular junctions, cytoskeletal organization, and intracellular trafficking as well as lipid metabolism and ion transport were among the most affected gene classes. Effects on several genes already identified as being part of vitamin D signaling in other cell types were observed along with genes known to affect insulin release, although with our assay we were not able to detect any effects of 1,25(OH)2D3 on glucose-stimulated insulin release from healthy pancreatic islets. CONCLUSION: The effects of 1,25(OH)2D3 on the expression of cytoskeletal and intracellular trafficking genes along with genes involved in ion transport may influence insulin exocytosis. However, an effect of 1,25(OH)2D3 on insulin release could not be detected for healthy islets in contrast to islets subjected to pathological conditions such as cytokine exposure and vitamin D deficiency as suggested by other studies. Thus, in addition to previously identified tolerogenic effects on the immune system, 1,25(OH)2D3 may affect basic functions of pancreatic beta cells, with the potential to render them more resistant to the detrimental conditions encountered during type 1 and 2 diabetes. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Deletion of C/EBP homologous protein (Chop) in C57Bl/6 mice dissociates obesity from insulin resistance.

AIMS/HYPOTHESIS: Endoplasmic reticulum (ER) stress has been implicated in the development of type 2 diabetes, via effects on obesity, insulin resistance and pancreatic beta cell health. C/EBP homologous protein (CHOP) is induced by ER stress and has a central role in apoptotic execution pathways triggered by ER stress. The aim of this study was to characterise the role of CHOP in obesity and insulin resistance. METHODS: Metabolic studies were performed in Chop ( -/- ) and wild-type C57Bl/6 mice, and included euglycaemic-hyperinsulinaemic clamps and indirect calorimetry. The inflammatory state of liver and adipose tissue was determined by quantitative RT-PCR, immunohistology and macrophage cultures. Viability and absence of ER stress in islets of Langerhans was determined by electron microscopy, islet culture and quantitative RT-PCR. RESULTS: Systemic deletion of Chop induced abdominal obesity and hepatic steatosis. Despite marked obesity, Chop ( -/- ) mice had preserved normal glucose tolerance and insulin sensitivity. This discrepancy was accompanied by lower levels of pro-inflammatory cytokines and less infiltration of immune cells into fat and liver. CONCLUSIONS/INTERPRETATION: These observations suggest that insulin resistance is not induced by fat accumulation per se, but rather by the inflammation induced by ectopic fat. CHOP may play a key role in the crosstalk between excessive fat deposition and induction of inflammation-mediated insulin resistance.

Platelet Gs hypofunction and abnormal morphology resulting from a heterozygous RGS2 mutation.

SUMMARY BACKGROUND: Regulator of G-protein signaling (RGS) 2 negatively regulates Gs signaling by inhibiting the activation of adenylyl cyclase (AC). RGS2 mRNA contains four translation initiation sites, leading to four isoforms with different abilities to inhibit AC activity; the largest isoform is the most pronounced inhibitor. A role for RGS2 in platelets is not known. OBJECTIVE: To describe a heterozygous RGS2 mutation (G23D) in three related patients, leading to Gs hypofunction in their platelets, and to study the mechanism behind the effect of the RGS2 mutation on platelet function and morphology. METHODS: Gs signaling was studied ex vivo in platelets and in vitro in transfected cells. Translation initiation was evaluated in vitro, and the interaction of wild-type and G23D RGS2 with AC was unraveled via immunoprecipitation. Platelet granule content was analyzed with proteomics. RESULTS: The mutation leads to reduced cAMP production after stimulation of Gs-coupled receptors. The largest RGS2 isoforms, with strong AC inhibitor activity, are enriched when the mutation is present, as compared with wild-type RGS2. Moreover, the mutation results in a stronger interaction of RGS2 with AC. G23D RGS2 carriers have enlarged, round platelets with abnormal alpha-granules. Proteomics of the platelet releasate revealed altered expression of some proteins involved in actin assembly, and carriers seemed to have a reduced platelet shape change. CONCLUSIONS: We present the first platelet Gs signaling defect caused by a heterozygous RGS2 variant that results in a unique mutational mechanism, such as the differential use of translation initiation sites resulting in different functional RGS2 isoforms.

Interferon regulatory factor-1 is a key transcription factor in murine beta cells under immune attack.

AIMS/HYPOTHESIS: IFN-gamma, together with other inflammatory cytokines such as IL-1beta and TNF-alpha, contributes to beta cell death in type 1 diabetes. We analysed the role of the transcription factor interferon regulatory factor (IRF)-1, a downstream target of IFN-gamma/signal transducer and activator of transcription (STAT)-1, in immune-mediated beta cell destruction. METHODS: Islets from mice lacking Irf-1 (Irf-1 (-/-)) and control C57BL/6 mice were transplanted in overtly diabetic NOD mice. Viability and functionality of islets were evaluated in vitro. Chemokine expression by Irf-1 (-/-) islets and INS-1E cells transfected with Irf-1 short interfering RNA (siRNA) was measured by real-time PCR as well as in functional assays in vitro. RESULTS: IRF-1 deletion in islets was associated with higher prevalence of primary non-function (63% vs 25%, p

Cluster analysis of rat pancreatic islet gene mRNA levels after culture in low-, intermediate- and high-glucose concentrations.

AIMS/HYPOTHESIS: Survival and function of insulin-secreting pancreatic beta cells are markedly altered by changes in nutrient availability. In vitro, culture in 10 rather than 2 mmol/l glucose improves rodent beta cell survival and function, whereas glucose concentrations above 10 mmol/l are deleterious. METHODS: To identify the mechanisms of such beta cell plasticity, we tested the effects of 18 h culture at 2, 5, 10 and 30 mmol/l glucose on the transcriptome of rat islets pre-cultured for 1 week at 10 mmol/l glucose using Affymetrix Rat 230 2.0 arrays. RESULTS: Culture in either 2-5 or 30 mmol/l instead of 10 mmol/l glucose markedly impaired beta cell function, while little affecting cell survival. Of about 16,000 probe-sets reliably detected in islets, some 5,000 were significantly up- or downregulated at least 1.4-fold by glucose. Analysis of these probe-sets with GeneCluster software identified ten mRNA profiles with unidirectional up- or downregulation between 2 and 10, 2 and 30, 5 and 10, 5 and 30 or 10 and 30 mmol/l glucose. It also identified eight complex V-shaped or inverse V-shaped profiles with a nadir or peak level of expression in 5 or 10 mmol/l glucose. Analysis of genes belonging to these various clusters using Onto-express and GenMAPP software revealed several signalling and metabolic pathways that may contribute to induction of beta cell dysfunction and apoptosis after culture in low- or high- vs intermediate-glucose concentration. CONCLUSIONS/INTERPRETATION: We have identified 18 distinct mRNA profiles of glucose-induced changes in islet gene mRNA levels that should help understand the mechanisms by which glucose affects beta cell survival and function under states of chronic hypo- or hyperglycaemia.

The development of atopic dermatitis is independent of Immunoglobulin E up-regulation in the K14-IL-4 SKH1 transgenic mouse model.

BACKGROUND: We have successfully generated an IgE-associated (extrinsic/allergic) mouse model of atopic dermatitis in K14-IL-4-Tg/CByB6 mice. The newly described subset of non-IgE-associated (intrinsic/non-allergic) atopic dermatitis in human patients raises the question on the role of IgE in the pathogenesis. OBJECTIVE: The aim of this study was to develop a non-IgE-associated atopic dermatitis model in K14-IL-4-Tg/SKH1 mice. METHODS: K14-IL-4-Tg/CByB6 mice were crossed with SKH1 mice to produce K14-IL-4-Tg/SKH1 mice. Phenotypes of clinical and histological, cytokine expression in the skin lesions, and total serum IgE in K14-IL-4-Tg/CByB6 and K14-IL-4-Tg/SKH1 mice were compared. The CD40 and CD40L on T and B cells were also studied to differentiate their roles in IgE production. RESULTS: K14-IL-4-Tg/SKH1mice had a normal total serum IgE level and manifested a chronic inflammatory skin phenotype identical to that of K14-IL-4-Tg/CByB6 IgE-mediated mice in clinical morphology, histology, infiltration of mononuclear cells/eosinophils/mast cells, mast cell degranulation, and up-regulation of chronic lesional cytokine mRNA expression of IL-1 beta, IL-3, IL-4, IL-6, IL-10, IL-12, IL-13, IFN-gamma, TNF-alpha, and TNF-beta. We also found that the inability of CD4(+) T cells of the K14-IL-4-Tg/SKH1mice to up-regulate CD40L expression upon stimulation might account for their inability to up-regulate the IgE level. B cell abnormality was ruled out as CD19(+) B cells of K14-IL-4-Tg/SKH1 mice synthesized the same amount of IgE in vitro compared with K14-IL-4-Tg/CByB6 mice in the presence of IL-4 and soluble CD40L. Our studies further suggested that the defect of early growth response-1 in T cells might be responsible for the impaired CD40L up-regulation in K14-IL-4-Tg/SKH1 mice. CONCLUSION: K14-IL-4-Tg/SKH1 mice developed skin inflammation that resembled human intrinsic atopic dermatitis. Therefore, this model may be suitable to study the pathogenesis of intrinsic atopic dermatitis.

Immune regulation of 1alpha-hydroxylase in murine peritoneal macrophages: unravelling the IFNgamma pathway.

The activated form of vitamin D(3), 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), plays an important role in the immune system. Indeed, receptors for 1,25(OH)(2)D(3) are found on most immune cells, and 1alpha-hydroxylase, the enzyme responsible for final activation of vitamin D(3), is expressed by monocytes/macrophages, resulting in secretion of 1,25(OH)(2)D(3) after immune stimulation. We have previously shown that in murine peritoneal macrophages 1alpha-hydroxylase is highly regulated by immune signals such as IFNgamma and LPS. In the present study we made use of two different knock-out mouse models with disruptions in two key transcription factors in the IFNgamma-signalling cascade (STAT1alpha and IRF1), to evaluate their role in the regulation of 1alpha-hydroxylase. This was performed by culturing peritoneal macrophages from these knock-out mice in the presence of IFNgamma and LPS, and evaluating the impact of the absence of the respective transcription factors on 1alpha-hydroxylase mRNA expression by real-time RT-PCR. In addition also the mRNA expression profiles of the essential transcription factors STAT1alpha, IRF1 and C/EBPbeta were investigated. The data confirm a crucial role for STAT1alpha as well as for C/EBPbeta in the regulation of 1alpha-hydroxylase in monocytes.

Evaluation of airway inflammation by quantitative Th1/Th2 cytokine mRNA measurement in sputum of asthma patients.

BACKGROUND: Asthma is a chronic inflammatory disorder of the airways driven by T cell activation. Th2 cells and their cytokines are thought to play a role in the pathophysiology of allergic as well as non-allergic asthma. METHODS: Airway cells were obtained by sputum induction from 15 healthy and 39 asthmatic individuals and the airway T cell cytokine profiles (interleukin (IL)-4, IL-5, IL-13, IL-10 and interferon (IFN)-gamma) at the mRNA level were studied by real time RT-PCR. RESULTS: Asthma patients had increased expression of IL-5 (p = 0.001) and IL-13 (p = 0.03) mRNA in sputum compared with non-asthmatic controls. IL-4 mRNA and IFN-gamma mRNA were detectable in the sputum of 44% and 21% of patients, respectively, but not in controls. Sputum IL-10 mRNA levels did not differ significantly between patients and controls. Sputum mRNA expression levels of IL-4, IL-5, and IL-13 were significantly correlated with the percentage of eosinophils and were higher in subjects with allergic asthma than in those with non-allergic asthma (p = 0.03, p = 0.02 and p = 0.0002, respectively); they did not differ between mild asthmatic subjects and those with moderate to severe asthma. In contrast, IFN-gamma mRNA expression was higher in non-allergic than in allergic patients (p = 0.04) and higher in patients with moderate to severe asthma than in those with mild asthma (p<0.01). Sputum IL-5 mRNA levels (but not the other cytokine mRNA levels) were also correlated with exhaled nitric oxide (eNO) and with bronchial hyperreactivity expressed as the histamine concentration resulting in a 20% decrease in forced expiratory volume in 1 second. CONCLUSION: Real time RT-PCR analysis of mRNA in induced sputum confirms a predominance of Th2 cytokines in both allergic and non-allergic asthma. IL-5 levels reflect eosinophil infiltration as well as eNO levels and hyperreactivity, and levels of the Th1 cytokine IFN-gamma indicate asthma severity. The technique is a promising tool for use in further studies of asthma severity and disease activity.

Involvement of 4-1BB (CD137)-4-1BBligand interaction in the modulation of CD4 T cell-mediated inflammatory colitis.

4-1BB ligand (4-1BBL) expressed on antigen-presenting cells interacts with 4-1BB on activated T cells (especially CD8+ cells) and co-stimulates the latter to secrete cytokines and to proliferate. The role of 4-1BB-4-1BBL interaction was studied here in a model of colitis based on naive CD4+ T cell transfer to SCID mice, a disease model in which CD8 cells do not take part. We found that CD4+ T cells from 4-1BB-deficient mice, after transfer in SCID mice, proliferated more rapidly compared to wild-type CD4+ T cells. Mice reconstituted with naive CD4+ T cells from 4-1BB-deficient mice developed colitis, however, with a mixed Th1/Th2 response, in contrast to the Th1-type response in mice reconstituted with wild-type naive CD4+ T cells. Importantly, this altered cytokine response did not temper colitis severity. Although it has been reported previously that 4-1BB co-stimulation may contribute to regulatory T cell functioning, we found that CD4+CD25+ regulatory T cells from 4-1BB-deficient mice were perfectly able to prevent naive CD4+ T cell-induced colitis. In conclusion, our data provide evidence that 4-1BB-4-1BBL interaction modulates the effector CD4+ T cell-driven immune response and cytokine production in experimental colitis without affecting regulatory T cell function.

The disease progression in the keratin 14 IL-4-transgenic mouse model of atopic dermatitis parallels the up-regulation of B cell activation molecules, proliferation and surface and serum IgE.

We have previously characterized the keratin 14 interleukin-4-transgenic (IL-4-Tg) mouse model of atopic dermatitis as a chronic pruritic inflammatory skin disease typified by skin infiltration of inflammatory cells and early up-regulation of Th2 cytokines and late surge of Th1 cytokines. In the present study, we examined the involvement of B cells. Systematic examinations of the following immunological parameters on B cells were carried out in non-Tg control mice and in IL-4-Tg mice at before disease onset and early and late disease stages so that we could determine the immunological sequence of events leading to the disease development: surface expressions of IA/IE, activation and costimulatory molecules, proliferation under LPS or IgM stimulation, quantification of cell surface and serum IgE, IgG1, and IgG2a. Our results showed that as the disease progresses from before onset to early disease and to late disease, there is a parallel increase in surface markers of B cell activation (IA/IE, CD44, CD69, CD80 and CD86), in B cell proliferation, and in cell surface and serum IgE. Significant increases of Th2-driven serum IgG1 and IgE in early disease was followed by significant increase of Th1-driven IgG2a in late disease. Importantly the significant increases of activation molecule (IA/IE), proliferation (to LPS), and surface IgE on B cells of the IL-4-Tg mice precedes the up-regulation of serum IgE and disease onset. These data suggest that activated B cells may play a role in atopic dermatitis disease development by up-regulating serum IgE concentration, which serves as a marker of disease onset.

Validation of real-time RT-PCR assays for mRNA quantification in baboons.

Real-time RT-PCR has been used widely, both in fundamental research and in clinical diagnostics, for instance for quantification of RNA levels in human tissues and tissue biopsies. In the present study we provide a strategy to validate primers/probes for real-time RT-PCR quantification of baboon samples. The method is based on the TaqMan system and uses primers/probes that have been designed and validated for human real-time RT-PCR. A prerequisite for the accuracy of this strategy is a similar amplification efficiency between human and baboon PCR reactions. We propose two different methods, i.e. by calculating PCR efficiencies from the slope of a dilution curve or by using the linear regression method, to compare the amplification efficiency between human and baboon samples. In conclusion, by performing a simple validation experiment, real-time PCR assays based on human sequences, which are easily available, can be applied for analysis of baboon samples.

1Alpha,25-dihydroxyvitamin D3 restores thymocyte apoptosis sensitivity in non-obese diabetic (NOD) mice through dendritic cells.

AIMS/HYPOTHESIS: Resistance of NOD thymocytes to apoptosis-inducing signals is restored by 1alpha,25-dihydroxyvitamin D3 (1alpha,25OH2D3), a therapy preventing diabetes in NOD mice. We studied whether modulation of thymocyte apoptosis is due to direct effects on thymic T lymphocytes or indirect effects via thymic dendritic cells, since both cell types constitute known targets for 1alpha,25OH2D3. METHODS AND RESULTS: Female NOD mice were treated with 1alpha,25OH2D3 (5microg/kg/2d) from 21 to 70 days. Vehicle-treated NOD and NOR mice served as controls. Analysis of thymic T lymphocytes from 1alpha,25OH2D3)-treated mice revealed a decrease in number of apoptosis-resistant CD4+CD8+ and CD4+CD8-HSA(high) T lymphocyte subsets, higher pro-apoptotic IL-2 and FasL, and lower anti-apoptotic Bclx-L mRNA expression levels. Thymic dendritic cells from 1alpha,25OH2D3-treated NOD mice had increased CD8alpha+FasL+ and CD80+/86+ expression compared to control NOD mice. In a syngeneic co-culture system of thymocytes and thymic dendritic cells, apoptosis levels were 20% higher only in co-cultures where both T cell- and dendritic cell-compartments originated from 1alpha,25OH2D3-treated mice. Activation-induced cell death-sensitivity in peripheral T lymphocytes was comparable to levels present in NOR mice, confirming better thymic selection in 1alpha,25OH2D3-treated mice. CONCLUSION/INTERPRETATION: We conclude that 1alpha,25OH2D3 needs both thymic T cell- and dendritic cell-compartments to exert its apoptosis-restorative effects in NOD thymocytes.

Early up-regulation of Th2 cytokines and late surge of Th1 cytokines in an atopic dermatitis model.

We investigated cytokine profiles in interleukin (IL)-4 transgenic (Tg) mice with a skin inflammatory disease resembling human atopic dermatitis. cDNA microarray revealed that the mRNAs encoding IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12p40, IL-13, tumour necrosis factor (TNF)-alpha, TNF-beta and interferon (IFN)-gamma were up-regulated in the skin of late lesion Tg mice and to a lesser degree in non-lesion Tg mice when compared to those of non-Tg mice. Real time reverse transcription-polymerase chain reaction (RT-PCR) analyses indicated that the cDNA copy numbers of IL-1beta, IL-4, IL-6, IL-10, TNF-alpha and IFN-gamma from the skin of late, early and non-lesions increased significantly compared to non-Tg mice. IL-2 and IL-12p40 cDNA copy numbers were increased significantly in early, but not late, lesions. Interestingly, IL-1beta, IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, TNF-alpha, and IFN-gamma cDNAs were increased significantly the skin of before-onset and/or non-lesion mice. Flow cytometry analyses demonstrated an increased percentage of keratinocytes producing IL-4 as the disease progressed. The percentage of IL-2, IL-4, IL-10 and IFN-gamma-producing T cells and IL-12-producing antigen-presenting cells in skin-draining lymph nodes and inflammatory skin also increased, particularly in mice with late lesion. These results suggest that disease induction is primarily triggered by Th2 cytokines and that Th1, Th2 and non-Th proinflammatory cytokines are all involved in the disease process.

NOD macrophages produce high levels of inflammatory cytokines upon encounter of apoptotic or necrotic cells.

During the development of type 1 diabetes, pancreatic beta-cells are subject to an immune attack, leading to their apoptotic or necrotic cell death. Apoptotic beta-cells are also present during periods of tissue remodeling, such as in early life. Macrophages should clear apoptotic cells silently without production of pro-inflammatory cytokines. The aim of the present study was to investigate the cytokine pattern of NOD macrophages exposed to apoptotic or necrotic cells in vitro. In contrast to the limited response of macrophages from C57BL/6 or NOR mice, NOD macrophages reacted aberrantly to both necrotic and apoptotic cells, with secretion of inappropriately high amounts of IL1beta and TNFalpha. Further exploration of the macrophage behavior showed an excessive response of NOD macrophages when exposed to LPS (high iNOS and IL12p40 levels), accompanied by hyper-activation of NF-kappaB(p65). In contrast, NOD macrophages failed to up-regulate IL1beta and IL12p40 in response to IFNgamma. This failure correlated with low protein levels and a low phosphorylation state of STAT1alpha. We conclude that NOD macrophages have severely aberrant cytokine expression patterns that could contribute to the initiation or continuation of an immune attack towards the pancreatic beta-cells and thus onset and progression of type 1 diabetes.

Vitamin D deficiency in early life accelerates Type 1 diabetes in non-obese diabetic mice.

AIMS/HYPOTHESIS: 1,25-dihydroxyvitamin D(3), the active form of vitamin D, prevents Type 1 diabetes in non-obese diabetic (NOD) mice. Epidemiological data show a threefold increase in human Type 1 diabetes when vitamin D deficiency was present in the first months of life. To evaluate whether a similar dietary deficiency affects diabetes incidence in NOD mice, we generated NOD mice with vitamin D deficiency in early life. METHODS: Breeding pairs of NOD mice, as well as their offspring (test mice), were kept in surroundings devoid of ultraviolet light and were fed a vitamin D-depleted diet for 100 days. Mice were followed for 250 days. RESULTS: At 250 days, 35% (12/35) male and 66% (22/33) female vitamin D-deficient mice were diabetic compared to 15% (6/40, p=0.05) and 45% (13/29, p<0.01) of the control mice. At 100 days no difference in insulitis was seen, but more vitamin D-deficient mice were glucose intolerant. Higher IL1 expression was detected in islets of vitamin D-deficient mice and their peritoneal macrophages had an aberrant cytokine profile (low IL1 and IL6, high IL15). Thymus and lymph nodes of vitamin D-deficient mice contained less CD4(+)CD62L(+) cells. CONCLUSION/INTERPRETATION: Vitamin D status increases the expression of Type 1 diabetes in NOD mice. Our data in NOD mice, as well as human epidemiological data, point to the importance of preventing vitamin D deficiency in early childhood. Controlling this dietary factor could be an easy and safe way to reduce the incidence of Type 1 diabetes in subjects who are genetically at risk.

The use of real-time reverse transcriptase PCR for the quantification of cytokine gene expression.

Real-time reverse transcriptase polymerase chain reaction (RT-PCR) is becoming a widely used method to quantify cytokines from cells, tissues, or tissue biopsies. The method allows for the direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in a single step. Using TaqMan chemistry (Applied Biosystems, Foster City, CA) and the ABI Prism 7700 Sequence Detection System (Applied Biosystems), we validated a large panel of murine and human cytokines, as we as other factors playing a role in the immune system, such a chemokines and apoptotic markers. Although the method allows fast, sensitive, and accurate quantification, different control assays are necessary for the method to be reliable. By construction of complementary DNA (cDNA) plasmid clones, standard curves are generated that allow direct quantification of every unknown sample. Furthermore, the choice of a reliable housekeeping gene is very important. Finally, co-amplification of contaminating genomic DNA is avoided by designing sets of primers located in different exons or or intron-exon junctions. In conclusion, the real-time RT-PCF technique is very accurate and sensitive, allows high through put, and can be performed on very small samples. The development of real-time RT-PCR has resulted in an exponential increase in its use over the last couple of years, and the method has undoubtedly become the standard for quantifying cytokine patterns, clarifying many functional properties of immune cells and their associated diseases.