Dendritic cells and macrophages are professional APCs that play a central role in initiating immune responses, linking innate and adaptive immunity. Chemerin is a novel chemoattractant factor that specifically attracts APCs through its receptor ChemR23. Interestingly, chemerin is secreted as a precursor of low biological activity, prochemerin, which upon proteolytic removal of a C-terminal peptide, is converted into a potent and highly specific agonist of its receptor. Given the fact that APCs are often preceded by polymorphonuclear cells (PMN) in inflammatory infiltrates, we hypothesized that PMN could mediate chemerin generation. We demonstrate here that human degranulated PMNs release proteases that efficiently convert prochemerin into active chemerin. The use of specific protease inhibitors allowed us to identify the neutrophil serine proteases cathepsin G and elastase as responsible for this process. Mass spectrometry analysis of processed prochemerin showed that each protease generates specifically a distinct form of active chemerin, differing in their C terminus and initially identified in human inflammatory fluids. These findings strongly suggest that bioactive chemerin generation takes place during the early stages of inflammation, underscoring the functional contribution of chemerin as a bridge between innate and adaptive immunity.

Chemokine receptors constitute an attractive family of drug targets in the frame of inflammatory diseases. However, targeting specific chemokine receptors may be complicated by their ability to form dimers or higher order oligomers. Using a combination of luminescence complementation and bioluminescence resonance energy transfer assays, we demonstrate for the first time the existence of hetero-oligomeric complexes composed of at least three chemokine receptors (CCR2, CCR5, and CXCR4). We show in T cells and monocytes that negative binding cooperativity takes place between the binding pockets of these receptors, demonstrating their functional interaction in leukocytes. We also show that specific antagonists of one receptor (TAK-779 or AMD3100) lead to functional cross-inhibition of the others. Finally, using the air pouch model in mice, we show that the CCR2 and CCR5 antagonist TAK-779 inhibits cell recruitment promoted by the CXCR4 agonist SDF-1␣, demonstrating that cross-inhibition by antagonists also occurs in vivo. Thus, antagonists of the therapeutically important chemokine receptors regulate the functional properties of other receptors to which they do not bind directly with important implications for the use of these agents in vivo.Chemokines are small chemoattractant cytokines that control a wide variety of biological and pathological processes, ranging from immunosurveillance to inflammation and from viral infection to cancer (1, 2). They mediate their effects by binding to cell surface receptors, which belong to the large family of G protein-coupled receptors (GPCRs). 4 Receptor binding initiates a cascade of intracellular events mediated by the association of the receptor with a heterotrimeric G protein, promoting guanine nucleotide exchange in the ␣ subunit and activation of the G protein (3). The G proteins trigger various effector enzymes, leading to chemotaxis and the regulation of a wide range of other functions, which vary in different cell populations. Because of their key role in inflammatory diseases, chemokines and their receptors constitute an attractive family of drug targets. However, the transfer of therapeutic compounds to clinical use has been hampered by the complexity and the functional redundancy of the chemokine system (4). The recent demonstration that chemokine receptors form both homo-and heteromers brings an additional layer of complexity to this system (reviewed in Ref. 5). There is therefore a need for a better understanding of how chemokine receptors are organized and regulated at the supramolecular level in the plasma membrane of primary leukocytes and how this organization affects the activity of agonists and antagonists of these receptors and their subsequent intracellular signaling network. Homodimerization has been reported for four chemokine receptors: CCR2, CCR5, CXCR2, and CXCR4 (6 -12). A recent report suggests that CXCR4 can also form constitutive homooligomers composed of at least three protomers (13). Heteromerization has been demonstrated between CCR2 and CCR5 or...

Development and stabilization of a vascular plexus requires the coordination of multiple signalling processes. Wnt planar cell polarity (PCP) signalling is critical in vertebrates for diverse morphogenesis events, which coordinate cell orientation within a tissue-specific plane. However, its functional role in vascular morphogenesis is not well understood. Here we identify PDZRN3, an ubiquitin ligase, and report that Pdzrn3 deficiency impairs embryonic angiogenic remodelling and postnatal retinal vascular patterning, with a loss of two-dimensional polarized orientation of the intermediate retinal plexus. Using in vitro and ex vivo Pdzrn3 loss-of-function and gain-of-function experiments, we demonstrate a key role of PDZRN3 in endothelial cell directional and coordinated extension. PDZRN3 ubiquitinates Dishevelled 3 (Dvl3), to promote endocytosis of the Frizzled/Dvl3 complex, for PCP signal transduction. These results highlight the role of PDZRN3 to direct Wnt PCP signalling, and broadly implicate this pathway in the planar orientation and highly branched organization of vascular plexuses.

Chemerin is a potent chemotactic factor that was identified recently as the ligand of ChemR23, a G protein-coupled receptor expressed by mononuclear phagocytes, dendritic cells (DCs), and NK cells. Chemerin is synthesized as a secreted precursor, prochemerin, which is poorly active on ChemR23. However, prochemerin can be converted rapidly into a full ChemR23 agonist by proteolytic removal of a carboxy-terminal peptide. This maturation step is mediated by the neutrophil-derived serine proteases elastase and cathepsin G. In the present work, we have investigated proteolytic events that negatively control chemerin activity. We demonstrate here that neutrophil-derived proteinase 3 (PR3) and mast cell (MC) chymase are involved in the generation of specific chemerin variants, which are inactive, as they do not induce calcium release or DC chemotaxis. Mass spectrometry analysis showed that PR3 specifically converts prochemerin into a chemerin form, lacking the last eight carboxy-terminal amino acids, and is inactive on ChemR23. Whereas PR3 had no effect on bioactive chemerin, MC chymase was shown to abolish chemerin activity by the removal of additional amino acids from its C-terminus. This effect was shown to be specific to bioactive chemerin (chemerin-157 and to a lesser extent, chemerin-156), as MC chymase does not use prochemerin as a substrate. These mechanisms, leading to the production of inactive variants of chemerin, starting from the precursor or the active variants, highlight the complex interplay of proteases regulating the bioactivity of this novel mediator during early innate immune responses.

The T cell integrin receptor LFA-1 orchestrates adhesion between T cells and antigen-presenting cells (APCs), resulting in formation of a contact zone known as the immune synapse (IS) which is supported by the cytoskeleton. L-plastin is a leukocyte-specific actin bundling protein that rapidly redistributes to the immune synapse following T cell-APC engagement. We used single domain antibodies (nanobodies, derived from camelid heavy-chain only antibodies) directed against functional and structural modules of L-plastin to investigate its contribution to formation of an immune synapse between Raji cells and human peripheral blood mononuclear cells or Jurkat T cells. Nanobodies that interact either with the EF hands or the actin binding domains of L-plastin both trapped L-plastin in an inactive conformation, causing perturbation of IS formation, MTOC docking towards the plasma membrane, T cell proliferation and IL-2 secretion. Both nanobodies delayed Ser(5) phosphorylation of L-plastin which is required for enhanced bundling activity. Moreover, one nanobody delayed LFA-1 phosphorylation, reduced the association between LFA-1 and L-plastin and prevented LFA-1 enrichment at the IS. Our findings reveal subtle mechanistic details that are difficult to attain by conventional means and show that L-plastin contributes to immune synapse formation at distinct echelons.