Functional Inactivation of CXC Chemokine Receptor 4–mediated Responses through SOCS3 Up-regulation

Hematopoietic cell growth, differentiation, and chemotactic responses require coordinated action between cytokines and chemokines. Cytokines promote receptor oligomerization, followed by Janus kinase (JAK) kinase activation, signal transducers and transactivators of transcription (STAT) nuclear translocation, and transcription of cytokine-responsive genes. These include genes that encode a family of negative regulators of cytokine signaling, the suppressors of cytokine signaling (SOCS) proteins. After binding their specific receptors, chemokines trigger receptor dimerization and activate the JAK/STAT pathway. We show that SOCS3 overexpression or up-regulation, stimulated by a cytokine such as growth hormone, impairs the response to CXCL12, measured by Ca2+ flux and chemotaxis in vitro and in vivo. This effect is mediated by SOCS3 binding to the CXC chemokine receptor 4 receptor, blocking JAK/STAT and Gαi pathways, without interfering with cell surface chemokine receptor expression. The data provide clear evidence for signaling cross-talk between cytokine and chemokine responses in building a functional immune system

Análisis de los residuos de los receptores de quimioquinas implicados en su dimerización y en la unión a Janus quinasas: nuevas dianas terapéuticas para alterar la funcionalidad de las quimioquinas

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 16-12-2005The chemokines are a large family of proteins that regulate leukocyte recruitment to inflammation sites and coordinate immune cell trafticking throughout the body. Chemokines mediate leukocyte function by binding to and activating specific G protein-coupled receptors (GPCR) expressed by these cell populations. 1t has been described that chemokine receptor dimerization plays an important role in signaling alter ligand binding. Chemokines trigger cell responses by stabilizing receptor dimerization and association of Janus kinases (JAK) to the receptor. JAK are then activated by transphosphorylation and phosphorylate the receptor, allowing G i activation and induction of the events that determine the final cell response. Bioinformatic analyses predicted that 11e52 in the CCR5 chemokine receptor transmembrane region-1 (TM1) and Va1150 in TM4 are key residues in the interaction surface between CCR5 molecules. Mutation of these residues generated non-functional receptors that did not dimerize or trigger signaling, In vitro and in vivo studies in human cell fines and primary T cells showed that synthetic peptides containing these residues blocked the responses induced by the CCR5 ligand. CCL5. Fluorescence resonance energy transfer (FRET) techniques showed the presence of preforrned, ligand-stabilized chemokine receptor oligomers. This is the first description of residues involved in chemokine receptor dimerization, and indicates a potential target for modification of chemokine responses. JAK proteins are constitutively associated with many cytokine receptors. but this association takes place in chemokine receptors only alter ligand binding. Although the cytokine region responsible for JAK binding has been described, the chemokine receptor domain involved in JAK binding remained unknown. Using hCCR2 as a model, we identified the domains involved in JAK2 binding to this receptor. We found that Leu 140 and Ala 141 in the CCR2 second intracellular loop were essential for JAK association. By mutating these residues, we generated CCR2 variants that showed no alterations in ligand binding affinity, but did not bind JAK2. These mutants were unable to signa', indicating that JAK association is crucial for chemokine function. Through a combination of in silico analysis, imaging techniques, biochemistry, and in vitro and in vivo functional analysis, we identified amino acid residues that are potential targets in the design of drugs that that affect chemokine responses specifically. 1

Syk-dependent ERK Activation Regulates IL-2 and IL-10 Production by DC Stimulated with Zymosan

Zymosan is a particulate yeast preparation that elicits high levels of IL-2 and IL-10 from dendritic cells (DC) and engages multiple innate receptors, including the Syk-coupled receptor dectin-1 and the MyD88-coupled receptor TLR2. Here, we show that induction of IL-2 and IL-10 by zymosan requires activation of ERK MAP kinase in murine DC. Surprisingly, ERK activation in response to zymosan is completely blocked in Syk-deficient DC and unaffected by MyD88 deficiency. Conversely, ERK activation in response to the TLR2 agonist Pam3Cys is completely MyD88 dependent and unaffected by Syk deficiency. The inability of TLR2 ligands in zymosan to couple to ERK may explain the Syk dependence of the IL-2 and IL-10 response in DC and emphasises the importance of Syk-coupled pattern recognition receptors such as dectin-1 in the detection of yeasts. Furthermore, the lack of receptor compensation observed here suggests that responses induced by complex innate stimuli cannot always be predicted by the signalling pathways downstream of individual receptors

Identification of a dendritic cell receptor that couples sensing of necrosis to immunity.

Injury or impaired clearance of apoptotic cells leads to the pathological accumulation of necrotic corpses, which induce an inflammatory response that initiates tissue repair. In addition, antigens present in necrotic cells can sometimes provoke a specific immune response and it has been argued that necrosis could explain adaptive immunity in seemingly infection-free situations, such as after allograft transplantation or in spontaneous and therapy-induced tumour rejection. In the mouse, the CD8alpha+ subset of dendritic cells phagocytoses dead cell remnants and cross-primes CD8+ T cells against cell-associated antigens. Here we show that CD8alpha+ dendritic cells use CLEC9A (also known as DNGR-1), a recently-characterized C-type lectin, to recognize a preformed signal that is exposed on necrotic cells. Loss or blockade of CLEC9A does not impair the uptake of necrotic cell material by CD8+ dendritic cells, but specifically reduces cross-presentation of dead-cell-associated antigens in vitro and decreases the immunogenicity of necrotic cells in vivo. The function of CLEC9A requires a key tyrosine residue in its intracellular tail that allows the recruitment and activation of the tyrosine kinase SYK, which is also essential for cross-presentation of dead-cell-associated antigens. Thus, CLEC9A functions as a SYK-coupled C-type lectin receptor to mediate sensing of necrosis by the principal dendritic-cell subset involved in regulating cross-priming to cell-associated antigens.This work was funded by Cancer Research UK. D.S. was supported by an EMBO long-term fellowship (ALTF 336-2004) and by a Marie Curie Intra-European Fellowship within the 6th European Community Framework Programme (MEIF-CT-2005-009205). We thank Edina Schweighoffer and Victor Tybulewicz for fetal liver from syk−/− embryos. We are grateful to members of the Immunobiology Laboratory, Cancer Research UK, for advice and discussions and the Biological Resources staff for animal care and assistance with mouse experiments.S

A framework for computational and experimental methods: indetifying dimreization residues in CCR chemokine receptors

Trabajo presentado en la European Conference for Computational Biology, celebrada en Madrid del 28 de septiembre al 1 de octubre de 2005.N

Blocking HIV-1 infection via CCR5 and CXCR4 receptors by acting in trans on the CCR2 chemokine receptor

The identification of chemokine receptors as HIV-1 coreceptors has focused research on developing strategies to prevent HIV-1 infection. We generated CCR2-01, a CCR2 receptor-specific monoclonal antibody that neither competes with the chemokine CCL2 for binding nor triggers signaling, but nonetheless blocks replication of monotropic (R5) and T-tropic (X4) HIV-1 strains. This effect is explained by the ability of CCR2-01 to induce oligomerization of CCR2 with the CCR5 or CXCR4 viral coreceptors. HIV-1 infection through CCR5 and CXCR4 receptors can thus be prevented in the absence of steric hindrance or receptor downregulation by acting in trans on a receptor that is rarely used by the virus to infect cells

Hetero-oligomerization of CCR2, CCR5, and CXCR4 and the Protean Effects of “Selective” Antagonists*

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-1α, 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