Suppressor of cytokine signalling-3 expression inhibits cytokine-mediated destruction of primary mouse and rat pancreatic islets and delays allograft rejection

AIMS/HYPOTHESIS: The pro-inflammatory cytokines IL-1 and IFNgamma are critical molecules in immune-mediated beta cell destruction leading to type 1 diabetes mellitus. Suppressor of cytokine signalling (SOCS)-3 inhibits the cytokine-mediated destruction of insulinoma-1 cells. Here we investigate the effect of SOCS3 in primary rodent beta cells and diabetic animal models. METHODS: Using mice with beta cell-specific Socs3 expression and a Socs3-encoding adenovirus construct, we characterised the protective effect of SOCS3 in mouse and rat islets subjected to cytokine stimulation. In transplantation studies of NOD mice and alloxan-treated mice the survival of Socs3 transgenic islets was investigated. RESULTS: Socs3 transgenic islets showed significant resistance to cytokine-induced apoptosis and impaired insulin release. Neither glucose-stimulated insulin release, insulin content or glucose oxidation were affected by SOCS3. Rat islet cultures transduced with Socs3-adenovirus displayed reduced cytokine-induced nitric oxide and apoptosis associated with inhibition of the IL-1-induced nuclear factor-kappaB and mitogen-activated protein kinase (MAPK) pathways. Transplanted Socs3 transgenic islets were not protected in diabetic NOD mice, but showed a prolonged graft survival when transplanted into diabetic allogenic BALB/c mice. CONCLUSIONS/INTERPRETATION: SOCS3 inhibits IL-1-induced signalling through the nuclear factor-kappaB and MAPK pathways and apoptosis induced by cytokines in primary beta cells. Moreover, Socs3 transgenic islets are protected in an allogenic transplantation model. SOCS3 may represent a target for pharmacological or genetic engineering in islet transplantation for treatment of type 1 diabetes mellitus.     

Cytokine-induced osteoprotegerin expression protects pancreatic beta cells through p38 mitogen-activated protein kinase signalling against cell death

Aims/hypothesis Pro-inflammatory cytokines play a crucial role in immune-mediated beta cell destruction, an essential mechanism in the pathogenesis of type 1 diabetes mellitus. Microarray analysis recently identified osteoprotegerin (OPG; now known as tumour necrosis factor receptor superfamily, member 11b [TNFRSF11B]) as a cytokine-induced gene in beta cells. The aim of the present study was to characterise the functional role and signalling pathways of OPG that are involved in cytokine-induced beta cell death. Materials and methods As cellular models, the rat beta cell line INS-1E and human primary pancreatic islets were employed. The effects of IL-1β and TNF-α on OPG expression were characterised by northern blot and immunoassay. The effect of OPG on beta cell survival was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Signalling pathways were evaluated by western blot analysis using antibodies against p38 mitogen-activated protein kinases (MAPK), c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2. Results The INS-1E cell line and primary pancreatic islets expressed OPG mRNA and secreted OPG protein, both of which were enhanced by IL-1β and TNF-α. Exposure to IL-1β resulted in sustained phosphorylation of p38 MAPK in INS-1E cells and subsequent cell death. Administration of exogenous OPG prevented both IL-1β-induced beta cell death and sustained p38 MAPK phosphorylation. Conclusions/interpretation Our data indicate that cytokine-induced production of OPG may protect beta cells from further damage. This protective effect is, at least in part, mediated through inhibition of p38 MAPK phosphorylation. Thus OPG is an autocrine or paracrine survival factor for beta cells.     

利拉鲁肽通过抑制核因子κB p65磷酸化调控人脐静脉内皮细胞一氧化氮合酶的表达

目的 探讨利拉鲁肽在高糖环境下对人脐静脉内皮细胞（HUVECs）功能的影响及其可能机制.方法 高糖环境下（25 mmol/L葡萄糖）培养人脐静脉内皮细胞,分别给予10、100、1 000 μg/L利拉鲁肽进行干预,采用实时定量聚合酶链反应（RT-PCR）和Western blotting法分别检测内皮型一氧化氮合酶（eNOS）、诱导型一氧化氮合酶（iNOS）以及核因子κB p65 （NF-κB p65）的mRNA、蛋白表达水平;应用肿瘤坏死因子-α（TNF-α）干预利拉鲁肽处理后的HUVECs,观察eNOS、iNOS、NF-κB p65的mRNA、蛋白表达水平的变化.研究结果多组间采用单因素方差分析,两组间比较采用SLD分析.结果 与普通培养基（7 mmol/L葡萄糖）组相比,高糖环境下HUVECs的eNOS mRNA和蛋白表达均降低,iNOS mRNA和蛋白表达及磷酸化NF-κB p65蛋白表达均增高（t=2.79、5.75、4.32、4.85、7.12,均P＜0.05）.与0μg/L组相比,1 000μg/L利拉鲁肽干预组HUVECs的eNOS mRNA、eNOS蛋白表达均上调,iNOS mRNA、iNOS蛋白表达及NF-κB p65磷酸化蛋白表达均下调（t=5.12、9.34、6.70、5.50、8.94,均P＜0.05）.与单独使用利拉鲁肽组相比,TNF-α联合利拉鲁肽组HUVECs的eNOSmRNA和蛋白表达均下调,iNOS mRNA和蛋白表达及磷酸化NF-κB p65蛋白表达均上调（t=3.33～7.87,均P＜0.05）.结论 利拉鲁肽可通过抑制NF-κB p65磷酸化在转录和翻译水平上增加eNOS表达、降低iNOS的表达,从而改善内皮细胞功能,预防糖尿病动脉粥样硬化.     

Nitric oxide contributes to cytokine-induced apoptosis in pancreatic beta cells via potentiation of JNK activity and inhibition of Akt

Aims/hypothesis Pro-inflammatory cytokines cause beta cell secretory dysfunction and apoptosis—a process implicated in the pathogenesis of type 1 diabetes. Cytokines induce the expression of inducible nitric oxide (NO) synthase (iNOS) leading to NO production. NO contributes to cytokine-induced apoptosis, but the underlying mechanisms are unclear. The aim of this study was to investigate whether NO modulates signalling via mitogen-activated protein kinases (MAPKs) and Akt.Materials and methods MAPK activities in INS-1 cells and isolated islets were determined by immunoblotting and in vitro kinase assay. Apoptosis was determined by ELISA measurement of histone–DNA complexes present in cytoplasm.Results Apoptosis in INS-1 cells induced by IL-1 plus IFN was dependent on NO production as demonstrated by the use of the NOS blocker N^G-methyl-l-arginine. Accordingly, an NO donor (S-nitroso-N-acetyl-d,l-penicillamine, SNAP) dose-dependently caused apoptosis in INS-1 cells. SNAP activated c-Jun N-terminal kinase (JNK) and p38 MAPK, but suppressed the activity of extracellular signal-regulated kinase MAPK. In rat islets, NOS inhibition decreased JNK and p38 activities induced by a 6-h exposure to IL-1. Likewise, IL-1-induced JNK and p38 activities were lower in iNOS^(–/–) mouse islets than in wild-type islets. In human islets, SNAP potentiated IL-1-induced JNK activation. The constitutive level of active, Ser473-phosphorylated Akt in INS-1 cells was suppressed by SNAP. IGF-I activated Akt and protected against SNAP-induced apoptosis. The anti-apoptotic effect of IGF-I was not associated with reduced JNK activation.Conclusions/interpretation We suggest that NO contributes to cytokine-induced apoptosis via potentiation of JNK activity and suppression of Akt.     

Beta cell death in hyperglycaemic Psammomys obesus is not cytokine-mediated

Aims/hypothesis It has recently been proposed that IL-1β may be responsible for beta cell death in type 2 diabetes mellitus. Major support for this assumption was derived from experiments in the gerbil Psammomys obesus (sand rat), a model for nutritionally induced non-insulin-dependent type 2 diabetes. Using gerbil-specific primers for the analysis of gene expression, we investigated the validity of this hypothesis.Methods Gene expression was analysed by real-time RT-PCR of isolated and laser-microdissected islets and by in situ RT-PCR, both in beta cells and in immune cells, as well as in lymph nodes and spleen.Results We were unable to detect Il-1β and the IL-1β-inducible enzyme inducible nitric oxide synthase (iNos) by in situ RT-PCR, either in the pancreatic beta cells, or in the small number of non-activated immune cells of healthy and diabetic Psammomys obesus after 1 and 3 weeks on a high-energy diet. Very low levels of Il-1β and iNos mRNA were detectable in collagenase-isolated and laser-microdissected islets of normoglycaemic gerbils by real-time RT-PCR without any increase of these mRNAs in islets from diabetic animals. These results were confirmed by electron microscopy with immunogold staining for IL-1β and insulin.Conclusions/interpretation The diabetic syndrome induced in Psammomys obesus by high-energy diet is a classical type 2 diabetes model, which does not show any evidence of an involvement of the proinflammatory cytokine IL-1β or of activated immune cells in its pathogenesis. This is clearly at variance with the situation in type 1 diabetes.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible to authorised users.     

Selective loss of peripheral blood CD45RO+ T lymphocytes correlates with increased levels of serum cytokines and endothelial cell-derived soluble cell adhesion molecules in patients with chronic idiopathic neutropenia of adults

 The present study was designed to investigate the hypothesis that selective loss of peripheral blood CD45RO⁺ T lymphocytes in patients with chronic idiopathic neutropenia of adults (CINA), previously reported from our laboratory, may be due to enhanced extravasation into the tissues. Serum levels of endothelial cell-derived soluble cell adhesion molecules (sELAM, sICAM and sVCAM), usually used as indicators of endothelial cell activation, were measured in 73 CINA patients and 32 healthy volunteers using a micro-ELISA method. We found that patients had markedly elevated concentrations of all three soluble cell adhesion molecules studied compared to the controls, and serum levels of sELAM, sICAM and, more importantly, sVCAM correlated inversely with the numbers of both CD4⁺/CD45RO⁺ and CD8⁺/CD45RO⁺ T cell subsets. Using a micro-ELISA method, we also measured serum levels of two endothelial cell activators, interleukin (IL)-1β and TNF-α, and found that CINA patients had significantly higher cytokine concentrations than control subjects. Serum levels of IL-1β and TNF-α correlated positively with the values of all three soluble cell adhesion molecules and inversely with the numbers of CD4⁺/CD45RO⁺ and CD8⁺/CD45RO⁺ T cell subsets. Moreover, we measured serum levels of the chemokine RANTES by a micro-ELISA technique and found that CINA patients also had elevated concentrations of the molecule compared to controls. Serum RANTES correlated positively with IL-1β, TNF-α, sICAM, sVCAM and sELAM and inversely with the numbers of both CD4⁺/CD45RO⁺ and CD8⁺/CD45RO⁺ T cell subsets. These findings strongly suggest that CINA patients have an activated endothelium to which CD45RA⁺ and CD45RO⁺ T cells tether and roll, but firm adhesion and transendothelial migration are restricted to CD45RO⁺ T cell subsets, as endothelial VCAM-1 interacts with the vascular leukocyte adhesion molecule-4 (VLA-4) constitutively expressed on CD45RO⁺ but not on CD45RA⁺ T cells. Subsequent subendothelial and tissue migration of CD45RO⁺ T cells may be facilitated by the chemokine RANTES, which acts mainly on CD45RO⁺ T cells. We concluded that selective loss of peripheral blood CD45RO⁺ T lymphocytes in CINA patients is probably due, at least in part, to enhanced extravasation of both CD4⁺/CD45RO⁺ and CD8⁺/CD45RO⁺ T cell subsets into the tissues. Received: February 18, 1998 / Accepted: June 10, 1998     

Glomerular expression of thrombospondin-1, transforming growth factor beta and connective tissue growth factor at different stages of diabetic nephropathy and their interdependent roles in mesangial response to diabetic stimuli

Aims/hypothesis We quantified the glomerular expression of thrombospondin-1 (THBS1, also known as TSP-1), transforming growth factor beta 1 (TGFB1, also known as TGF-β1) and connective tissue growth factor (CTGF) at each stage of diabetic nephropathy. We also examined the roles of THBS1 and CTGF in mediating high-glucose- and glycated-albumin-induced synthesis of the matrix protein, fibronectin, by mesangial cells. Methods THBS1, latent and active TGFB1, and CTGF, were detected by immunohistochemistry and in situ hybridisation in biopsies from 19 insulin-dependent diabetic patients with incipient, manifest and advanced diabetic nephropathy, and in 11 control kidneys. Findings were quantified by image analysis. Human mesangial cells were cultured with normal or high glucose, albumin or glycated albumin (Amadori product), +/−THBS1 or CTGF antisense oligonucleotides, or with peptide W, an inhibitor of TGFB1 bioactivation by THBS1. Proteins were measured by western blot analysis or ELISA. Results In glomeruli of normal kidneys, mRNA and protein levels for THBS1, latent-TGFB1 and CTGF were low. They were increased in the incipient stage of diabetic nephropathy, predominantly in mesangial areas, with further increases at later stages of the disease. Little or no active TGFB1 immunostaining was detected prior to manifest diabetic nephropathy. In contrast to high-glucose conditions, increases in fibronectin synthesis that were stimulated by glycated albumin were not dependent on THBS1 activation of latent TGFB1. However, increased fibronectin synthesis in both conditions required CTGF. Conclusions/interpretation Increased glomerular expression of all three factors occurs from the earliest stage of diabetic nephropathy. In contrast to THBS1, CTGF is required for mesangial synthesis of fibronectin stimulated by high glucose or glycated albumin, and is thus a potential therapeutic target. N. A. Wahab and L. Schaefer made an equal contribution to the work reported in this paper     

Cytokines activate genes of the endocytotic pathway in insulin-producing RINm5F cells

Aims/hypothesis Cytokines are important humoral mediators of beta cell destruction in autoimmune diabetes. The aim of this study was to identify novel cytokine-induced genes in insulin-producing RINm5F cells, which may contribute to beta cell death or survival.Methods A global gene expression profile in cytokine-exposed insulin-producing RINm5F cells was achieved by automated restriction fragment differential display PCR. The expression of selected candidate genes was confirmed by real-time RT-PCR analysis.Results Exposure of RINm5F cells to IL-1 or to a cytokine mixture (IL-1, TNF-, IFN-) for 6 h resulted in the differential expression of a functional gene cluster. Apart from the well-known up-regulation of the cytokine-responsive genes iNOS, NF-B, MnSOD and Hsp70, several genes that belong to the functional cluster of the endocytotic pathway were identified. These endocytotic genes comprised: clathrin, megalin, synaptotagmin and calcineurin, which were up-regulated by IL-1 or the cytokine mixture. In contrast, the expression of the calcineurin inhibitor CAIN and of the GDP/GTP exchange protein Rab3 was down-regulated by cytokines. Other up-regulated cytokine-responsive genes were: agrin, murine adherent macrophage protein mRNA (MAMA) and transport-associated protein (TAP1/MTP), whereas the plasma membrane calcium ATPase (PMCA) 2 and PMCA 3 genes were down-regulated by cytokines.Conclusions/interpretation Our results indicate that genes of the endocytotic pathway are regulated by pro-inflammatory cytokines. This might affect the density of cytokine receptors at the beta cell surface and concomitantly the sensitivity of the cells to cytokine toxicity. A better understanding of the functional cross-talk between endocytotic and cytokine signalling pathways could further the development of novel strategies to protect pancreatic beta cells against toxic effects of pro-inflammatory cytokines.Electronic Supplementary Material Electronic supplementary material is available in the online version of this article at Abbreviations CAIN calcineurin inhibitor - EP extension-protected adaptor - Hsp70 heat shock protein 70 - iNOS inducible nitric oxide synthase - MAMA murine adherent macrophage protein - MnSOD manganese superoxide dismutase - NF-B nuclear factor kappa B - NO nitric oxide - PMCA plasma membrane calcium ATPase - Rab3 rab3 GDP/GTP exchange protein - RFDD-PCR restriction fragment differential display PCR - SD standard adaptor - Syt synaptotagmin V - TAP1 transport-associated protein 1     

The role of low-grade inflammation in the polycystic ovary syndrome

PCOS is not only the most frequent cause of oligomenorrhea in young women, but also a metabolic disorder characterized by insulin resistance, glucose intolerance, dyslipidemia, and obesity, especially the visceral phenotype. PCOS represents a broad spectrum of endocrine and metabolic alterations which change with age and with increasing adiposity. In fact, during adolescence and youth the predominant clinical manifestations of PCOS are menstrual abnormalities, hirsutism and acne, whereas in peri-menopausal and post-menopausal periods metabolic disorders and an increased risk for cardiovascular diseases prevail. The pathogenetic links between PCOS and metabolic or cardiovascular complications are still debated. However, recent evidence has been focused on a condition of low-grade chronic inflammation as a potential cause of the long-term consequence of the syndrome.