TLR3 and Rig-like receptor on myeloid dendritic cells and Rig-like receptor on human NK cells are both mandatory for production of IFN-gamma in response to double-stranded RNA.

Cross-talk between NK cells and dendritic cells (DCs) is critical for the potent therapeutic response to dsRNA, but the receptors involved remained controversial. We show in this paper that two dsRNAs, polyadenylic-polyuridylic acid and polyinosinic-polycytidylic acid [poly(I:C)], similarly engaged human TLR3, whereas only poly(I:C) triggered human RIG-I and MDA5. Both dsRNA enhanced NK cell activation within PBMCs but only poly(I:C) induced IFN-gamma. Although myeloid DCs (mDCs) were required for NK cell activation, induction of cytolytic potential and IFN-gamma production did not require contact with mDCs but was dependent on type I IFN and IL-12, respectively. Poly(I:C) but not polyadenylic-polyuridylic acid synergized with mDC-derived IL-12 for IFN-gamma production by acting directly on NK cells. Finally, the requirement of both TLR3 and Rig-like receptor (RLR) on mDCs and RLRs but not TLR3 on NK cells for IFN-gamma production was demonstrated using TLR3- and Cardif-deficient mice and human RIG-I-specific activator. Thus, we report the requirement of cotriggering TLR3 and RLR on mDCs and RLRs on NK cells for a pathogen product to induce potent innate cell activation.

The nuclear hormone receptor PPARγ counteracts vascular calcification by inhibiting Wnt5a signalling in vascular smooth muscle cells.

Vascular calcification is a hallmark of advanced atherosclerosis. Here we show that deletion of the nuclear receptor PPARγ in vascular smooth muscle cells of low density lipoprotein receptor (LDLr)-deficient mice fed an atherogenic diet high in cholesterol, accelerates vascular calcification with chondrogenic metaplasia within the lesions. Vascular calcification in the absence of PPARγ requires expression of the transmembrane receptor LDLr-related protein-1 in vascular smooth muscle cells. LDLr-related protein-1 promotes a previously unknown Wnt5a-dependent prochondrogenic pathway. We show that PPARγ protects against vascular calcification by inducing the expression of secreted frizzled-related protein-2, which functions as a Wnt5a antagonist. Targeting this signalling pathway may have clinical implications in the context of common complications of atherosclerosis, including coronary artery calcification and valvular sclerosis.

Prostaglandin E(2) promotes colorectal adenoma growth via transactivation of the nuclear peroxisome proliferator-activated receptor delta.

Cyclooxygenase-derived prostaglandin E(2) (PGE(2)) is the predominant prostanoid found in most colorectal cancers (CRC) and is known to promote colon carcinoma growth and invasion. However, the key downstream signaling pathways necessary for PGE(2)-induced intestinal carcinogenesis are unclear. Here we report that PGE(2) indirectly transactivates PPARdelta through PI3K/Akt signaling, which promotes cell survival and intestinal adenoma formation. We also found that PGE(2) treatment of Apc(min) mice dramatically increased intestinal adenoma burden, which was negated in Apc(min) mice lacking PPARdelta. We demonstrate that PPARdelta is a focal point of crosstalk between the prostaglandin and Wnt signaling pathways which results in a shift from cell death to cell survival, leading to increased tumor growth.

T cell receptor-ligand interactions: a conformational preequilibrium or an induced fit.

Kinetic parameters of T cell receptor (TCR) interactions with its ligand have been proposed to control T cell activation. Analysis of kinetic data obtained has so far produced conflicting insights; here, we offer a consideration of this problem. As a model system, association and dissociation of a soluble TCR (sT1) and its specific ligand, an azidobenzoic acid derivative of the peptide SYIPSAEK-(ABA)I (residues 252-260 from Plasmodium berghei circumsporozoite protein), bound to class I MHC H-2K(d)-encoded molecule (MHCp) were studied by surface plasmon resonance. The association time courses exhibited biphasic patterns. The fast and dominant phase was assigned to ligand association with the major fraction of TCR molecules, whereas the slow component was attributed to the presence of traces of TCR dimers. The association rate constant derived for the fast phase, assuming a reversible, single-step reaction mechanism, was relatively slow and markedly temperature-dependent, decreasing from 7.0 x 10(3) at 25 degrees C to 1.8 x 10(2) M(-1).s(-1) at 4 degrees C. Hence, it is suggested that these observed slow rate constants are the result of unresolved elementary steps of the process. Indeed, our analysis of the kinetic data shows that the time courses of TCR-MHCp interaction fit well to two different, yet closely related mechanisms, where an induced fit or a preequilibrium of two unbound TCR conformers are operational. These mechanisms may provide a rationale for the reported conformational flexibility of the TCR and its unusual ligand recognition properties, which combine high specificity with considerable crossreactivity.

Role of endothelin receptor subtypes in volume-stimulated ANF secretion.

The role of endothelin (ET) receptors was tested in volume-stimulated atrial natriuretic factor (ANF) secretion in conscious rats. Mean ANF responses to slow infusions (3 x 3.3 ml/8 min) were dose dependently reduced (P < 0.05) by bosentan (nonselective ET-receptor antagonist) from 64.1 +/- 18.1 (SE) pg/ml (control) to 52.6 +/- 16.1 (0.033 mg bosentan/rat), 16.1 +/- 7.6 (0. 33 mg/rat), and 11.6 +/- 6.5 pg/ml (3.3 mg/rat). The ET-A-receptor antagonist BQ-123 (1 mg/rat) had no effect relative to DMSO controls, whereas the putative ET-B antagonist IRL-1038 (0.1 mg/rat) abolished the response. In a second protocol, BQ-123 (>/=0.5 mg/rat) nonsignificantly reduced the peak ANF response (106.1 +/- 23.0 pg/ml) to 74.0 +/- 20.5 pg/ml for slow infusions (3.5 ml/8.5 min) but reduced the peak response (425.3 +/- 58.1 pg/ml) for fast infusions (6.6 ml/1 min) by 49.9% (P < 0.001) and for 340 pmoles ET-1 (328.8 +/- 69.5 pg/ml) by 83.5% (P < 0.0001). BQ-123 abolished the ET-1-induced increase in arterial pressure (21.8 +/- 5.2 mmHg at 1 min). Changes in central venous pressure were similar for DMSO and BQ-123 (slow: 0.91 and 1.14 mmHg; fast: 4.50 and 4.13 mmHg). The results suggest 1) ET-B receptors mainly mediate the ANF secretion to slow volume expansions of <1.6%/min; and 2) ET-A receptors mainly mediate the ANF response to acute volume overloads.

Peroxisome proliferator-activated receptor beta/delta activation inhibits hypertrophy in neonatal rat cardiomyocytes.

OBJECTIVE: Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) is the predominant PPAR subtype in cardiac cells and plays a prominent role in the regulation of cardiac lipid metabolism. However, the role of PPARbeta/delta activators in cardiac hypertrophy is not yet known. METHODS AND RESULTS: In cultured neonatal rat cardiomyocytes, the selective PPARbeta/delta activator L-165041 (10 micromol/L) inhibited phenylephrine (PE)-induced protein synthesis ([(3)H]leucine uptake), induction of the fetal-type gene atrial natriuretic factor (ANF) and cardiac myocyte size. Induction of cardiac hypertrophy by PE stimulation also led to a reduction in the transcript levels of both muscle-type carnitine palmitoyltransferase (50%, P&lt;0.05) and pyruvatedehydrogenase kinase 4 (30%, P&lt;0.05), and these changes were reversed in the presence of the PPARbeta/delta agonist L-165041. Stimulation of neonatal rat cardiomyocytes with PE and embryonic rat heart-derived H9c2 cells with lipopolysaccharide (LPS) enhanced the expression of the nuclear factor (NF)-kappaB-target gene monocyte chemoattractant protein 1 (MCP-1). The induction of MCP-1 was reduced in the presence of L-165041, suggesting that this compound prevented NF-kappaB activation. Electrophoretic mobility shift assay (EMSA) revealed that L-165041 significantly decreased LPS-stimulated NF-kappaB binding activity in H9c2 myotubes. Finally, coimmunoprecipitation studies showed that L-165041 strongly enhanced the physical interaction between PPARbeta/delta and the p65 subunit of NF-kappaB, suggesting that increased association between these two proteins is the mechanism responsible for antagonizing NF-kappaB activation by PPARbeta/delta activators. CONCLUSION: These results suggest that PPARbeta/delta activation inhibits PE-induced cardiac hypertrophy and LPS-induced NF-kappaB activation.

The CD4-like molecule LAG-3, biology and therapeutic applications.

IMPORTANCE OF THE FIELD: Promising immunotherapeutic agents targeting co-stimulatory pathways are currently being tested in clinical trials. One player in this array of regulatory pathways is the LAG-3/MHC class II axis. The lymphocyte activation gene-3 (LAG-3) is a negative co-stimulatory receptor that modulates T cell homeostasis, proliferation and activation. A recombinant soluble dimeric form of LAG-3 (sLAG-3-Ig, IMP321) shows adjuvant properties and enhances immunogenicity of tumor vaccines. Recent clinical trials produced encouraging results, especially when the human dimeric soluble form of LAG-3 (hLAG-3-Ig) was used in combination with chemotherapy. AREAS COVERED IN THIS REVIEW: The biological relevance of LAG-3 in vivo. Pre-clinical data demonstrating adjuvant properties, as well as the improvement of tumor immunity by sLAG-3-Ig. Recent advances in the clinical development of the therapeutic reagent IMP321, hLAG-3-Ig, for cancer treatment. WHAT THE READER WILL GAIN: This review summarizes preclinical and clinical data on the biological functions of LAG-3. TAKE HOME MESSAGE: The LAG-3 inhibitory pathway is attracting attention, in the light of recent studies demonstrating its role in T cell unresponsiveness, and Treg function after chronic antigen stimulation. As a soluble recombinant dimer, the sLAG-3-Ig protein acts as an adjuvant for therapeutic induction of T cell responses, and may be beneficial to cancer patients when used in combination therapies.

Gene transfer of the Ly-3 chain gene of the mouse CD8 molecular complex: co-transfer with the Ly-2 polypeptide gene results in detectable cell surface expression of the Ly-3 antigenic determinants.

The CD8 molecule is a glycoprotein expressed on a subset of mature T lymphocytes. It has been postulated to be a receptor for class I major histocompatibility complex molecules. In the mouse, CD8 is a heterodimer composed of Ly-2 and Ly-3 chains. We have isolated and analyzed cDNA and cosmid clones corresponding to the Ly-3 subunit. One of the isolated, cosmid clones was subsequently transfected, alone or in combination with the Ly-2 gene, into mouse Ltk- cells. Analysis of the Ly-2,3 molecules expressed at the surface of the double transfectants indicated that they are serologically and biochemically indistinguishable from their normal counterparts expressed on lymphoid cells. Ltk- cells transfected with the Ly-2 gene alone were shown to react with a subset of anti-CD8 monoclonal antibodies whereas Ly-3 transfectants did not stain with any of the anti-Ly-3 antibodies employed in this study. Since at least one of these antibodies (53-5.8) has been previously shown to recognize an epitope which is retained on the Ly-3 subunit after dissociation of the heterodimeric Ly-2,3 complex, these observations suggest that the expression of the Ly-2 polypeptide is required to permit the detectable cell surface expression of the antigenic determinants carried by the Ly-3 subunit.

Gene transfer of a soluble IL-1 type 2 receptor-Ig fusion protein improves cardiac allograft survival in rats.

OBJECTIVE: Interleukin-1 (IL-1) mediates ischemia-reperfusion injury and graft inflammation after heart transplantation. IL-1 affects target cells through two distinct types of transmembrane receptors, type-1 receptor (IL-1R1), which transduces the signal, and the non-signaling type-2 receptor (IL-1R2), which acts as a ligand sink that subtracts IL-1beta from IL-1R1. We analyzed the efficacy of adenovirus (Ad)-mediated gene transfer of a soluble IL-1R2-Ig fusion protein in delaying cardiac allograft rejection and the mechanisms underlying the protective effect. METHODS: IL-1 inhibition by IL-1R2-Ig was tested using an in vitro functional assay whereby endothelial cells preincubated with AdIL-1R2-Ig or control virus were stimulated with recombinant IL-1beta or tumor necrosis factor-alpha (TNF-alpha), and urokinase-type plasminogen activator (u-PA) induction was measured by zymography. AdIL-1R2-Ig was delivered to F344 rat donor hearts ex vivo, which were placed in the abdominal position in LEW hosts. Intragraft inflammatory cell infiltrates and proinflammatory cytokine expression were analyzed by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. RESULTS: IL-1R2-Ig specifically inhibited IL-1beta-induced u-PA responses in vitro. IL-1R2-Ig gene transfer reduced intragraft monocytes/macrophages and CD4(+) cell infiltrates (p<0.05), TNF-alpha and transforming growth factor-beta (TGF-beta) expression (p<0.05), and prolonged graft survival (15.6+/-5.7 vs 10.3+/-2.5 days with control vector and 10.1+/-2.1 days with buffer alone; p<0.01). AdIL-1R2-Ig combined with a subtherapeutic regimen of cyclosporin A (CsA) was superior to CsA alone (19.4+/-3.0 vs 15.9+/-1.8 days; p<0.05). CONCLUSIONS: Soluble IL-1 type-2 receptor gene transfer attenuates cardiac allograft rejection in a rat model. IL-1 inhibition may be useful as an adjuvant therapy in heart transplantation.

Allorestricted T lymphocytes with a high avidity T-cell receptor towards NY-ESO-1 have potent anti-tumor activity.

The cancer-testis antigen NY-ESO-1 has been targeted as a tumor-associated antigen by immunotherapeutical strategies, such as cancer vaccines. The prerequisite for a T-cell-based therapy is the induction of T cells capable of recognizing the NY-ESO-1-expressing tumor cells. In this study, we generated human T lymphocytes directed against the immunodominant NY-ESO-1(157-165) epitope known to be naturally presented with HLA-A*0201. We succeeded to isolate autorestricted and allorestricted T lymphocytes with low, intermediate or high avidity TCRs against the NY-ESO-1 peptide. The avidity of the established CTL populations correlated with their capacity of lysing HLA-A2-positive, NY-ESO-1-expressing tumor cell lines derived from different origins, e.g. melanoma and myeloma. The allorestricted NY-ESO-1-specific T lymphocytes displayed TCRs with the highest avidity and best anti-tumor recognition activity. TCRs derived from allorestricted, NY-ESO-1-specific T cells may be useful reagents for redirecting primary T cells by TCR gene transfer and, therefore, may facilitate the development of adoptive transfer regimens based on TCR-transduced T cells for the treatment of NY-ESO-1-expressing hematological malignancies and solid tumors.

Expression of the glucagon-like peptide-1 receptor gene in rat brain.

Evidence that glucagon-like peptide-1 (GLP-1) (7-36) amide functions as a novel neuropeptide prompted us to study the gene expression of its receptor in rat brain. Northern blot analysis showed transcripts of similar size in RINm5F cells, hypothalamus, and brain-stem. First-strand cDNA was prepared by using RNA from hypothalamus, brainstem, and R1Nm5F cells and subsequently amplified by PCR. Southern blot analysis of the PCR products showed a major 1.4-kb band in all these preparations. PCR products amplified from hypothalamus were cloned, and the nucleotide sequence of one strand was identical to that described in rat pancreatic islets. In situ hybridization studies showed specific labeling in both neurons and glia of the thalamus, hypothalamus, hippocampus, primary olfactory cortex, choroid plexus, and pituitary gland. In the hypothalamus, ventromedial nuclei cells were highly labeled. These findings indicate that GLP-1 receptors are actually synthesized in rat brain. In addition, the colocalization of GLP-1 receptors, glucokinase, and GLUT-2 in the same areas supports the idea that these cells play an important role in glucose sensing in the brain.

The adaptor complex 2 directly interacts with the alpha 1b-adrenergic receptor and plays a role in receptor endocytosis.

Using the yeast two-hybrid system, we identified the mu 2 subunit of the clathrin adaptor complex 2 as a protein interacting with the C-tail of the alpha 1b-adrenergic receptor (AR). Direct association between the alpha 1b-AR and mu 2 was demonstrated using a solid phase overlay assay. The alpha 1b-AR/mu 2 interaction occurred inside the cells, as shown by the finding that the transfected alpha 1b-AR and the endogenous mu 2 could be coimmunoprecipitated from HEK-293 cell extracts. Mutational analysis of the alpha 1b-AR revealed that the binding site for mu 2 does not involve canonical YXX Phi or dileucine motifs but a stretch of eight arginines on the receptor C-tail. The binding domain of mu 2 for the receptor C-tail involves both its N terminus and the subdomain B of its C-terminal portion. The alpha 1b-AR specifically interacted with mu 2, but not with the mu 1, mu 3, or mu 4 subunits belonging to other AP complexes. The deletion of the mu 2 binding site in the C-tail markedly decreased agonist-induced receptor internalization as demonstrated by confocal microscopy as well as by the results of a surface receptor biotinylation assay. The direct association of the adaptor complex 2 with a G protein-coupled receptor has not been reported so far and might represent a common mechanism underlying clathrin-mediated receptor endocytosis.

Mice from a genetically resistant background lacking the interferon gamma receptor are susceptible to infection with Leishmania major but mount a polarized T helper cell 1-type CD4+ T cell response.

Mice with homologous disruption of the gene coding for the ligand-binding chain of the interferon (IFN) gamma receptor and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in the differentiation of functional CD4+ T cell subsets in vivo and resistance to infection. Wild-type 129/Sv/Ev mice are resistant to infection with this parasite, developing only small lesions, which resolve spontaneously within 6 wk. In contrast, mice lacking the IFN-gamma receptor develop large, progressing lesions. After infection, lymph nodes (LN) and spleens from both wild-type and knockout mice showed an expansion of CD4+ cells producing IFN-gamma as revealed by measuring IFN-gamma in supernatants of specifically stimulated CD4+ T cells, by enumerating IFN-gamma-producing T cells, and by Northern blot analysis of IFN-gamma transcripts. No biologically active interleukin (IL) 4 was detected in supernatants of in vitro-stimulated LN or spleen cells from infected wild-type or deficient mice. Reverse transcription polymerase chain reaction analysis with primers specific for IL-4 showed similar IL-4 message levels in LN from both types of mice. The IL-4 message levels observed were comparable to those found in similarly infected C57BL/6 mice and significantly lower than the levels found in BALB/c mice. Anti-IFN-gamma treatment of both types of mice failed to alter the pattern of cytokines produced after infection. These data show that even in the absence of IFN-gamma receptors, T helper cell (Th) 1-type responses still develop in genetically resistant mice with no evidence for the expansion of Th2 cells.

Expression of the mammalian Xenotropic Polytropic Virus Receptor 1 (XPR1) in tobacco leaves leads to phosphate export.

Phosphate homeostasis in multicellular eukaryotes depends on both phosphate influx and efflux. The mammalian Xenotropic Polytropic Virus Receptor 1 (XPR1) shares homology to the Arabidopsis PHO1, a phosphate exporter expressed in roots. However, phosphate export activity of XPR1 has not yet been demonstrated in a heterologous system. Here, wedemonstrate that transient expression in tobacco leaves of XPR1-GFP leads to specific phosphate export. Like PHO1-GFP, XPR1-GFP is localized predominantly to the endomembrane system in tobacco cells. These results show that tobacco leaves are a good heterologous system to study the transport activity of members of the PHO1/XPR1 family.