SummaryWe have shown previously that dendritic cells (DC) produce IL-12 upon interaction with CD4+ T cells. Here we ask how this IL-12 production is induced and regulated. Quantitative PCR. and in situ hybridization for IL-12 p40 and an ELISA specific for the p70 heterodimer were used to determine IL-12 production. We demonstrate that ligation of either CD40 or MHC class II molecules independently trigger IL-12 production in DC, and that IL-12 production is downregulated by IL-4 and IL-10. The levels ofbioactive IL-12 that can be released by triggering with an anti-CD40 mAb or with a T cell hybridoma are high (range 260-4700 pg/ml from 1 • 106 DC in 72 h). The CD40-mediated pathway indicates that IL-12 production is induced in DC upon interaction with activated, CD40 ligand-expressing helper T cells, even in the absence of cognate antigen recognition. Side-by-side comparison oflL-12 production, and blocking experiments employing an anti-CD40 ligand n'LAb, suggest that the CD40-mediated pathway is quantitatively more significant than induction via the MHC class II molecule. The importance of the CD40/CD40 ligand interaction for IL-12 induction in DC likely contributes to the recent finding that mice lacking the CD40 ligand are impaired in mounting Thl type cell-mediated immune responses. IL-12, a 70-kD heterodimeric cytokine composed of co-.valentty linked p35 and p40 chains has emerged as a central cytokine in the immune response (1). IL-12 stimulates NK cells, mediates Thl development, and fosters CTL development. It can be produced by monocytes and macrophages in response to intracellular pathogens, bacteria (e.g., staphylococci) and bacterial products. Recent reports indicate that dendritic cells (DC) also release bioactive IL-12. One report described that anti-IL-12 blocks the capacity of murine DC to skew the response of naive transgenic T cells to the Thl phenotype (2), and another shows induction of IL-12 p40/p35 mRNA in bone-marrow derived murine DC upon uptake ofmicroparticle-absorbed protein antigen (3). Human epidermal Langerhans cells are also a source of IL-12 (4). We have recently used several criteria for demonstration of IL-12 p40 and p35 mRNA as well as IL-12 p40 and bioactive p70 proteins, to show that murine and human DC release IL-12 upon conventional stimuli such as staphylococcus aureus (5). We also found that DC produced bioactive IL-12 upon interaction with T cells without standard stir " ~-,~h as bacterial products. Here, we describe the regulation oflL-12 in DC.

Interleukin-12 (IL-12), a 70-kDa heterodimeric cytokine composed of covalently linked p35 and p40 chains, is to date the most critical factor for skewing the immune response towards a T helper 1 (Th1) of cytokine profile [high interferon-gamma (IFN-gamma), low IL-4]. Established sources of IL-12 are stimulated macrophages, neutrophils and B cells. As dendritic cells (DC) process antigen in the periphery and then migrate to lymphoid organs to sensitize T cells and induce cell mediated immunity, we reasoned that DC should constitute a critical source of IL-12. The criteria used to detect IL-12 in DC were the demonstration of p40 and p35 mRNA (semiquantitative polymerase chain reaction, northern blotting, and in situ hybridization) as well as IL-12 protein (p70 enzyme-linked immunosorbent assay, p70 antigen capture followed by IFN-gamma bioassay, free p40 chain radioimmunoassay or immunoprecipitation). We found that conventional stimuli such as Staphylococcus aureus induced production of IL-12 by murine as well as human DC in amounts comparable to spleen cells, peritoneal macrophages or peripheral mononuclear cells. DC exhibited, however, features that had not been seen with other antigen-presenting cells: they produced bioactive IL-12 upon antigen-specific interaction with T cells without any other stimuli; in an allogeneic mixed leukocyte reaction model, neutralizing anti-IL-12 antibodies showed that DC-derived IL-12 was critical for optimal proliferation and IFN-gamma production by activated Th1 blasts; and finally, the priming of resting, naive allogeneic T cells by DC, followed by restimulation of primed T blasts by DC, skewed the response to Th1 without the need for any exogenous cytokines or stimuli such as microorganisms. This skewing to Th1 cytokine production, which depended on DC-derived IL-12, but did not require anti-IL-4, exogenous IL-12, or microbes, might be a major function of DC.

OBJECTIVETo assess the potential role of FoxP3-expressing regulatory T cells (Tregs) in reversing obesity-linked insulin resistance and diabetic nephropathy in rodent models and humans.RESEARCH DESIGN AND METHODSTo characterize the role of Tregs in insulin resistance, human visceral adipose tissue was first evaluated for Treg infiltration and second, the db/db mouse model was evaluated.RESULTSObese patients with insulin resistance displayed significantly decreased natural Tregs but an increase in adaptive Tregs in their visceral adipose tissue as compared with lean control subjects. To further evaluate the pathogenic role of Tregs in insulin resistance, the db/db mouse model was used. Treg depletion using an anti-CD25 monoclonal antibody enhanced insulin resistance as shown by increased fasting blood glucose levels as well as an impaired insulin sensitivity. Moreover, Treg-depleted db/db mice developed increased signs of diabetic nephropathy, such as albuminuria and glomerular hyperfiltration. This was paralleled by a proinflammatory milieu in both murine visceral adipose tissue and the kidney. Conversely, adoptive transfer of CD4+FoxP3+ Tregs significantly improved insulin sensitivity and diabetic nephropathy. Accordingly, there was increased mRNA expression of FoxP3 as well as less abundant proinflammatory CD8+CD69+ T cells in visceral adipose tissue and kidneys of Treg-treated animals.CONCLUSIONSData suggest a potential therapeutic value of Tregs to improve insulin resistance and end organ damage in type 2 diabetes by limiting the proinflammatory milieu.

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Background Secretoneurin is a neuropeptide located in nerve fibers along blood vessels, is up-regulated by hypoxia and induces angiogenesis. We tested the hypothesis that secretoneurin gene therapy exerts beneficial effects in a rat model of myocardial infarction and evaluated the mechanism of action on coronary endothelial cells. Methods and Results In-vivo secretoneurin improved left ventricular function, inhibited remodeling and reduced scar formation. In the infarct border zone secretoneurin induced coronary angiogenesis as shown by increased density of capillaries and arteries. In-vitro secretoneurin induced capillary tubes, stimulated proliferation, inhibited apoptosis and activated Akt and ERK in coronary endothelial cells. Effects were abrogated by a VEGF-antibody and secretoneurin stimulated VEGF receptors in these cells. Secretoneurin furthermore increased binding of VEGF to endothelial cells and binding was blocked by heparinase indicating that secretoneurin stimulates binding of VEGF to heparan sulfate proteoglycan binding sites. Additionally, secretoneurin increased binding of VEGF to its co-receptor neuropilin 1. In endothelial cells secretoneurin also stimulated FGF receptor-3 and IGF-1 receptor and in coronary vascular smooth muscle cells we observed stimulation of VEGF receptor-1 and FGF receptor-3. Exposure of cardiac myocytes to hypoxia and ischemic heart after myocardial infarction revealed increased secretoneurin m-RNA and protein. Conclusions Our data show that secretoneurin acts as an endogenous stimulator of VEGF signaling in coronary endothelial cells by enhancing binding of VEGF to low affinity binding sites and neuropilin 1 and stimulates further growth factor receptors like FGF receptor-3. Our in-vivo findings indicate that secretoneurin might be a promising therapeutic tool in ischemic heart disease.