Michel Detheux

Specific Recruitment of Antigen-presenting Cells by Chemerin, a Novel Processed Ligand from Human Inflammatory Fluids

Dendritic cells (DCs) and macrophages are professional antigen-presenting cells (APCs) that play key roles in both innate and adaptive immunity. ChemR23 is an orphan G protein–coupled receptor related to chemokine receptors, which is expressed specifically in these cell types. Here we present the characterization of chemerin, a novel chemoattractant protein, which acts through ChemR23 and is abundant in a diverse set of human inflammatory fluids. Chemerin is secreted as a precursor of low biological activity, which upon proteolytic cleavage of its COOH-terminal domain, is converted into a potent and highly specific agonist of ChemR23, the chemerin receptor. Activation of chemerin receptor results in intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of p42–p44 MAP kinases, through the Gi class of heterotrimeric G proteins. Chemerin is structurally and evolutionary related to the cathelicidin precursors (antibacterial peptides), cystatins (cysteine protease inhibitors), and kininogens. Chemerin was shown to promote calcium mobilization and chemotaxis of immature DCs and macrophages in a ChemR23-dependent manner. Therefore, chemerin appears as a potent chemoattractant protein of a novel class, which requires proteolytic activation and is specific for APCs.

The Second Extracellular Loop of CCR5 Is the Major Determinant of Ligand Specificity

The chemokine receptor CCR5 binds macrophage inflammatory protein (MIP)-1α, MIP-1β, and regulated on activation, normal T-cell expressed and secreted (RANTES), and constitutes the major co-receptor allowing infection of CD4+ T lymphocytes, macrophages, and microglial cells by macrophage-tropic strains of human and simian immunodeficiency virus. CCR5 is most closely related to CCR2b, another chemokine receptor that responds to monocyte chemoattractant protein (MCP)-1, MCP-2, MCP-3, and MCP-4. We have investigated by mutagenesis the regions of CCR5 and CCR2b involved in the specificity of binding and functional response to their respective ligands. We demonstrate that the key region of CCR5 involved in its specific interaction with MIP-1α, MIP-1β, and RANTES, and its subsequent activation, lies within the second extracellular loop (and possibly the adjacent transmembrane segments). Conversely, the NH2-terminal domain of CCR2b is responsible for the high affinity binding of MCP-1, but is not sufficient to confer activation of the intracellular cascades. Extracellular loops of the receptor, among which the second loop plays a prominent role, are necessary to achieve efficient signaling of the receptor. These data complement our previous mapping of CCR5 domains functionally involved in the fusion process with the human immunodeficiency virus envelope, and will help in the development of agents able to interfere with the early steps of viral infection.

The Orexin OX1 Receptor Activates a Novel Ca2+ Influx Pathway Necessary for Coupling to Phospholipase C

Ca2+ elevations in Chinese hamster ovary cells stably expressing OX1 receptors were measured using fluorescent Ca2+ indicators fura-2 and fluo-3. Stimulation with orexin-A led to pronounced Ca2+elevations with an EC50 around 1 nm. When the extracellular [Ca2+] was reduced to a submicromolar concentration, the EC50 was increased 100-fold. Similarly, the inositol 1,4,5-trisphosphate production in the presence of 1 mm external Ca2+ was about 2 orders of magnitude more sensitive to orexin-A stimulation than in low extracellular Ca2+. The shift in the potency was not caused by depletion of intracellular Ca2+ but by a requirement of extracellular Ca2+ for production of inositol 1,4,5-trisphosphate. Fura-2 experiments with the “Mn2+-quench technique” indicated a direct activation of a cation influx pathway by OX1 receptor independent of Ca2+ release or pool depletion. Furthermore, depolarization of the cells to +60 mV, which almost nullifies the driving force for Ca2+ entry, abolished the Ca2+ response to low concentrations of orexin-A. The results thus suggest that OX1 receptor activation leads to two responses, (i) a Ca2+ influx and (ii) a direct stimulation of phospholipase C, and that these two responses converge at the level of phospholipase C where the former markedly enhances the potency of the latter.

Pharmacological characterization of human NPFF1 and NPFF2 receptors expressed in CHO cells by using NPY Y1 receptor antagonists

Neuropeptide FF (NPFF) belongs to an opioid-modulatory system including two precursors (pro-NPFF(A) and pro-NPFF(B)) and two G-protein coupled receptors (NPFF(1) and NPFF(2)). The pharmacological and functional profiles of human NPFF(1) and NPFF(2) receptors expressed in Chinese hamster ovary (CHO) cells were compared by determining the affinity of several peptides derived from both NPFF precursors and by measuring their abilities to inhibit forskolin-induced cAMP accumulation. Each NPFF receptor recognizes peptides from both precursors with nanomolar affinities, however, with a slight preference of pro-NPFF(A) peptides for NPFF(2) receptors and of pro-NPFF(B) peptides for NPFF(1) receptors. BIBP3226 ((R)-N(2)-(diphenylacetyl)-N-[(4-hydroxyphenyl)-methyl]-argininamide) and BIBO3304 ((R)-N(2)-(diphenylacetyl)-N-[4-(aminocarbonylaminomethyl)-benzyl]-argininamide trifluoroacetate), two selective neuropeptide Y (NPY) Y(1) receptor antagonists, display relative high affinities for NPFF receptors and exhibit antagonist properties towards hNPFF(1) receptors. The structural determinants responsible for binding of these molecules to NPFF receptors were investigated and led to the synthesis of hNPFF(1) receptor antagonists with affinities from 40 to 80 nM. Our results demonstrate differences in pharmacological characteristics between NPFF(1) and NPFF(2) receptors and the feasibility of subtype-selective antagonists.

Functional characterization of a human receptor for neuropeptide FF and related peptides

Neuropeptides FF (NPFF) and AF (NPAF) are involved in pain modulation and opioid tolerance. These peptides were known to act through uncharacterized G protein‐coupled receptors (GPCR). We describe here, using an aequorin‐based assay as screening tool, that an orphan GPCR, previously designated HLWAR77, is a functional high affinity receptor for NPFF and related peptides. This receptor is further designated as NPFFR. Binding experiments were performed with a new radioiodinated probe, [125I]‐EYF, derived from the EFW‐NPSF sequence of the rat NPFF precursor. Chinese hamster ovary (CHO) cell membranes expressing NPFFR bound [125I]‐EYF with a Kd of 0.06u2003nM. Various NPFF analogues and related peptides inhibited [125I]‐EYF specific binding with the following rank order (Ki): human NPAF (0.22u2003nM), SQA‐NPFF (0.29u2003nM), NPFF (0.30u2003nM), 1DMe (0.31u2003nM), EYW‐NPSF (0.32u2003nM), QFW‐NPSF (0.35u2003nM), 3D (1.12u2003nM), Met‐enk‐RF‐NH2 (3.25u2003nM), FMRF‐NH2 (10.5u2003nM) and NPSF (12.1u2003nM). The stimulatory activity of the same set of peptides was measured by a functional assay based on the co‐expression of NPFFR, Gα16 and apoaequorin. The rank order of potency was consistent with the results of the binding assay. Membranes from NPFFR expressing CHO cells bound GTPγ[35S] in the presence of SQA‐NPFF. This functional response was prevented by pertussis toxin treatment, demonstrating the involvement of Gi family members. SQA‐NPFF inhibited forskolin induced cyclic AMP accumulation in recombinant CHO cells in a dose dependent manner. This response was abolished as well by pertussis toxin pre‐treatment. RTu2003–u2003PCR analysis of human tissues mRNA revealed that expression of NPFFR was mainly detected in placenta, thymus and at lower levels in pituitary gland, spleen and testis.

Adaptation of Aequorin Functional Assay to High Throughput Screening

AequoScreen™, a cellular aequorin-based functional assay, has been optimized for luminescent high-throughput screening (HTS) of G protein-coupled receptor (GPCRs). AequoScreen is a homogeneous assay in which the cells are loaded with the apoaequorin cofactor coelenterazine, diluted in assay buffer, and injected into plates containing the samples to be tested. A flash of light is emitted following the calcium increase resulting from the activation of the GPCR by the sample. Here we have validated a new plate reader, the Hamamatsu Photonics FDSS6000, for HTS in 96- and 384-well plates with CHO-K1 cells stably coexpressing mitochondrial apoaequorin and different GPCRs (AequoScreen cell lines). The acquisition time, plate type, and cell number per well have been optimized to obtain concentration-response curves with 4000 cells/well in 384-well plates and a high signal: background ratio. The FDSS6000 and AequoScreen cell lines allow reading of twenty 96- or 384-well plates in 1 h with Z’ values of 0.71 and 0.78, respectively. These results bring new insights to functional assays, and therefore reinforce the interest in aequorin-based assays in a HTS environment.

Identification and characterization of an endogenous chemotactic ligand specific for FPRL2

Chemotaxis of dendritic cells (DCs) and monocytes is a key step in the initiation of an adequate immune response. Formyl peptide receptor (FPR) and FPR-like receptor (FPRL)1, two G protein–coupled receptors belonging to the FPR family, play an essential role in host defense mechanisms against bacterial infection and in the regulation of inflammatory reactions. FPRL2, the third member of this structural family of chemoattractant receptors, is characterized by its specific expression on monocytes and DCs. Here, we present the isolation from a spleen extract and the functional characterization of F2L, a novel chemoattractant peptide acting specifically through FPRL2. F2L is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein, an intracellular tetrapyrolle-binding protein. The peptide binds and activates FPRL2 in the low nanomolar range, which triggers intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of extracellular signal–regulated kinase 1/2 mitogen-activated protein kinases through the Gi class of heterotrimeric G proteins. When tested on monocytes and monocyte-derived DCs, F2L promotes calcium mobilization and chemotaxis. Therefore, F2L appears as a new natural chemoattractant peptide for DCs and monocytes, and the first potent and specific agonist of FPRL2.

Agonist and antagonist activities on human NPFF2 receptors of the NPY ligands GR231118 and BIBP3226

Neuropeptide FF (NPFF) is a part of a neurotransmitter system acting as a modulator of endogenous opioid functions. At this time, no non‐peptide or peptide NPFF‐antagonists have been discovered. Here, we demonstrate that Neuropeptide Y (NPY) ligands, in fact possess significant ability to interact with the human NPFF2 receptors. NPY Y1 antagonist BIBP3226 and mixed Y1 antagonist/Y4 agonist GR231118 are able to displace with low affinity, 50u2003–u2003100u2003nM, the specific binding on NPFF receptors expressed in CHO cells as well as in rat dorsal spinal cord, an affinity however superior to those determined against Y2, Y4 or Y5 receptors. Furthermore, BIBP3226 which is unable to inhibit the forskolin‐stimulated cyclic AMP production mediated by NPFF2 receptors, antagonizes the effect of NPFF, revealing the first antagonist of NPFF receptors. These properties of NPY ligands on Neuropeptide FF receptors must be considered when evaluating pharmacological activities of these drugs.

Aequorin-Based Functional Assays for G-Protein-Coupled Receptors, Ion Channels, and Tyrosine Kinase Receptors

Aequorin is a photoprotein originating from jellyfish, whose luminescent activity is dependent on the concentration of calcium ions. Due to the high sensitivity and low background linked to luminescent assays, as well as to its absence of toxicity and its large linear dynamic range, aequorin has been used as an intracellular calcium indicator since its discovery in the early 1960s. The first applications of aequorin involved its microinjection in cells. The cloning of its gene in 1985 opened the way to the stable expression of aequorin in cell lines or even entire organisms. Here we present the validation of aequorin as a functional assay for the screening of G-protein-coupled receptors, ion channels, and tyrosine kinase receptors, as well as for their pharmacological characterization in agonist and antagonist detection assays. We optimized our cell suspension-based assay and determined that the most sensitive assay was performed at room temperature, with mitochondrially expressed aequorin and using coelenterazine derivative h for reconstitution of aequorin. The robustness of the assay and the current availability of luminometers with integrated injectors allow aequorin to fit perfectly with high throughput functional assays requirements.

Urokinase plasminogen activator and plasmin efficiently convert hemofiltrate CC chemokine 1 into its active.

We have previously isolated from human hemofiltrate an N-terminally truncated form of the hemofiltrate CC chemokine 1 (HCC-1), and characterized HCC-1[9–74] as a strong agonist of CCR1, CCR5, and to a lower extent CCR3. In this study, we show that conditioned media from human tumor cell lines PC-3 and 143B contain proteolytic activities that convert HCC-1 into the [9–74] form. This activity was fully inhibited by inhibitors of urokinase-type plasminogen activator (uPA), including PA inhibitor-1, an anti-uPA mAb, and amiloride. Pure preparations of uPA processed HCC-1 with high efficiency, without further degrading HCC-1[9–74]. Plasmin could also generate HCC-1[9–74], but degraded the active product as well. The kinetics of HCC-1 cleavage by uPA and plasmin (Michaelis constant, Km, of 0.76 ± 0.4 μM for uPA, and 0.096 ± 0.05 μM for plasmin; catalytic rate constant, kcat: 3.36 ± 0.96 s−1 for uPA and 6 ± 3.6 s−1 for plasmin) are fully compatible with a role in vivo. The activation of an abundant inactive precursor into a broad-spectrum chemokine by uPA and plasmin directly links the production of uPA by numerous tumors and their ability to recruit mononuclear leukocytes, without the need for the transcriptional activation of chemokine genes.