RESUMEN
CC chemokine receptor 4 (CCR4) is expressed by Th2 and regulatory T cells and directs their migration along gradients of the chemokines CCL17 and CCL22. Both chemokines and receptor are upregulated in allergic disease, making CCR4 a therapeutic target for the treatment of allergy. We set out to assess the mechanisms underlying a previous report that CCL22 is a dominant ligand of CCR4, which may have implications for its therapeutic targeting. Human T cells expressing endogenous CCR4 and transfectants engineered to express CCR4 were assessed for receptor function, using assays of calcium release, chemotaxis, receptor endocytosis, and ligand binding. Despite the two ligands having equal potency in calcium flux and chemotaxis assays, CCL22 showed dominance in both receptor endocytosis assays and heterologous competitive binding assays. Using two different CCR4-specific Abs, we showed that CCR4 exists in at least two distinct conformations, which are differentially activated by ligand. A major population is activated by both CCL17 and CCL22, whereas a minor population is activated only by CCL22. Mutation of a single C-terminal residue K310 within a putative CCR4 antagonist binding site ablated activation of CCR4 by CCL17, but not by CCL22, despite having no effect on the binding of either ligand. We conclude that CCL17 and CCL22 are conformationally selective ligands of CCR4 and interact with the receptor by substantially different mechanisms. This finding suggests that the selective blockade of CCR4 in allergy may be feasible when one CCR4 ligand dominates, allowing the inhibition of Th2 signaling via one ligand while sparing regulatory T cell recruitment via another.
Asunto(s)
Quimiotaxis de Leucocito/inmunología , Hipersensibilidad/inmunología , Receptores CCR4/inmunología , Linfocitos T/inmunología , Animales , Calcio/inmunología , Calcio/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/inmunología , Quimiocina CCL17/química , Quimiocina CCL17/inmunología , Quimiocina CCL22/química , Quimiocina CCL22/inmunología , Quimiocina CCL22/metabolismo , Quimiotaxis de Leucocito/genética , Endocitosis/inmunología , Citometría de Flujo , Humanos , Hipersensibilidad/genética , Hipersensibilidad/metabolismo , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Modelos Moleculares , Mutación , Unión Proteica/inmunología , Conformación Proteica , Estructura Terciaria de Proteína , Receptores CCR4/química , Receptores CCR4/genética , Linfocitos T/metabolismoRESUMEN
High-throughput screening of the corporate compound collection led to the discovery of a novel series of N-substituted-5-aryl-oxazolidinones as potent human CCR8 antagonists. The synthesis, structure-activity relationships, and optimization of the series that led to the identification of SB-649701 (1a), are described.
Asunto(s)
Oxazolidinonas/síntesis química , Oxazolidinonas/farmacología , Receptores de Quimiocina/antagonistas & inhibidores , Animales , Células CHO , Quimiotaxis de Leucocito/efectos de los fármacos , Simulación por Computador , Cricetinae , Cricetulus , Evaluación Preclínica de Medicamentos , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Humanos , Indicadores y Reactivos , Proteína Quinasa de Distrofia Miotónica , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Receptores CCR8 , Relación Estructura-Actividad , Células Th2/efectos de los fármacosRESUMEN
CCL1, the CCR8 ligand, is a CC chemokine secreted by activated monocytes and lymphocytes and is a potent chemoattractant for these cell types. The in vivo role of the CCL1/CCR8 axis in Th2-mediated inflammation is far from clear. Ligand neutralisation studies reported discrepancies in the effect of CCL1/CCR8 and CCR8 knockout studies showed very different insights into the functional role of the CCR8. To further study the biological function of CCL1, we focused on the generation and characterisation of RNA aptamers. We report here the in vitro isolation of the first nuclease resistant and selective RNA aptamer (T48) with high-binding affinity for human and mouse CCL1. The T48 aptamer but not a random control aptamer antagonises CCL1 function in a dose-dependent fashion in both heparin binding and chemotaxis assays. To our knowledge, the T48 aptamer constitutes one of the most potent CCL1 antagonists reported to date and is an excellent tool to dissect CCL1-specific function in vivo. The T48 aptamer may also have potential as new generation of therapeutic tools.
Asunto(s)
Bioquímica/métodos , Quimiocinas CC/química , ARN/química , Animales , Secuencia de Bases , Quimiocina CCL1 , Quimiocinas/química , Quimiocinas/metabolismo , Factores Quimiotácticos/química , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In Vitro , Inflamación , Ligandos , Ratones , Datos de Secuencia Molecular , Conformación de Ácido NucleicoRESUMEN
CXCL10 (also known as IP-10 in humans and CRG-2 in mice) is a nonglycosylated chemokine and a member of the non-ELR CXC chemokine subfamily implicated in a variety of inflammatory conditions. The role of CXCL10 in different disease states still requires clarification, and new approaches are necessary to better understand its biological function. We report here the isolation of a series of nuclease-resistant RNA aptamers that act to antagonize human CXCL10 function in a number of in vitro and cell-based assays. The two most potent aptamers identified were highly selective for human CXCL10. A further aptamer was identified that antagonized both the human and the mouse CXCL10. A combination of a molecular-biology-based truncation and solid-phase synthesis enabled the truncation of one of the aptamers from 71 to 34 nucleotides. This was followed by PEGylation, 3' capping, and further stabilization of the RNA aptamer, while its high potency was maintained. These aptamers could be utilized as powerful target validation tools and may also have therapeutic potential. To our knowledge, the CXCL10 aptamers generated are the most potent antagonists of CXCL10/CXCR3 signaling reported to date.
Asunto(s)
Inhibición de Migración Celular , Quimiocinas CXC/antagonistas & inhibidores , Quimiocinas CXC/biosíntesis , Interferón gamma/fisiología , ARN/química , Animales , Secuencia de Bases , Sitios de Unión/genética , Células CHO , Línea Celular Tumoral , Quimiocina CXCL10 , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Cricetinae , Humanos , Ligandos , Ratones , Datos de Secuencia Molecular , Polietilenglicoles/química , ARN/síntesis química , ARN/aislamiento & purificación , ARN/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ratas , Receptores CXCR3 , Receptores de Quimiocina/antagonistas & inhibidores , Receptores de Quimiocina/biosíntesis , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
The CC chemokine receptor 4 (CCR4) shows selectivity for the recruitment of memory T cell subsets, including those of the T helper cell type 2 (Th2) phenotype. In humans, CCR4+ T cells are recruited to the asthmatic lung in response to allergen challenge; however, the contribution of this pathway to allergic disease remains uncertain. We therefore investigated the role of CCR4 in allergic airways inflammation in the guinea pig. Blockade of CCR4 with a specific antibody resulted in only minor changes in numbers of CCR4+ Th cells in the bronchoalveolar lavage fluid of allergen-challenged guinea pigs and failed to inhibit the generation of eotaxin/CC chemokine ligand (CCL)11 or macrophage-derived chemokine/CCL22 or the recruitment of inflammatory leukocytes to the lung. These data suggest that although CCR4 was originally proposed as a marker of Th2 status, antigen-specific Th2 cells are recruited to the lung predominantly by other pathways. This study casts doubts on the validity of CCR4 as a therapeutic target in the treatment of asthma.
Asunto(s)
Asma/terapia , Receptores de Quimiocina/antagonistas & inhibidores , Animales , Asma/etiología , Movimiento Celular , Quimiocina CCL22 , Quimiocinas CC/biosíntesis , Cobayas , Pulmón/patología , Receptores CCR4 , Receptores de Quimiocina/fisiología , Linfocitos T/fisiología , Células Th2/fisiologíaRESUMEN
We studied the regulation of CCR4 expression in peripheral blood and in human models of cutaneous and pulmonary allergen challenge. CCR4 expression was detectable on freshly isolated CD4+ lymphocytes and in CD4+ and CD8+ T cell lines derived from blood of atopic donors. Numbers of CCR4+ cells were up-regulated in T cell lines expanded in the presence of IL-4. CCR4 mRNA was absent at baseline in normal subjects in lung and skin, but present at baseline in the lung of some atopics. Baseline expression of CCR4 mRNA and protein was higher in lung vs. skin, but allergen-induced increases in CCR4 mRNA+ cells were observed in both organs. CCR4 protein+ cells were present at higher levels after allergen challenge in atopics compared to normal subjects. CCR4 may be important in the recruitment of T lymphocytes at sites of allergic inflammation, in a non-organ-specific manner.
Asunto(s)
Asma/inmunología , Hipersensibilidad Inmediata/inmunología , Pulmón/inmunología , Receptores de Quimiocina/biosíntesis , Rinitis Alérgica Estacional/inmunología , Piel/inmunología , Adulto , Asma/sangre , Asma/patología , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular , Células Cultivadas , Quimiocina CCL17 , Quimiocina CCL22 , Quimiocinas CC/biosíntesis , Quimiotaxis de Leucocito/inmunología , Femenino , Expresión Génica , Humanos , Hipersensibilidad Inmediata/sangre , Hipersensibilidad Inmediata/patología , Ligandos , Masculino , Receptores CCR4 , Receptores de Quimiocina/genética , Rinitis Alérgica Estacional/sangre , Rinitis Alérgica Estacional/patologíaRESUMEN
Th2 lymphocytes play a central role in the control and maintenance of allergic inflammation. The chemokine receptor CCR4 is preferentially expressed on the surface of Th2 lymphocytes polarised in vitro. However, CCR4 is found on the surface of a significant proportion of circulating memory T lymphocytes, some of which are capable of producing the Th1-associated cytokine interferon gamma. To investigate the function of CCR4 on guinea pig (gp) T lymphocytes, we identified the open-reading frame of gpCCR4, which encodes a 361-amino acid protein with 88 and 81% amino acid identity to human and murine CCR4 sequences, respectively. Cells transfected with gpCCR4 migrated toward the human and murine orthologues of the CCR4 ligands, macrophage-derived chemokine and thymus and activation-regulated chemokine. Surface expression of CCR4, using an anti-human CCR4 monoclonal antibody, 10E4, was detected on approximately 12% of guinea pig peripheral blood T helper cells, and CCR4(+) guinea pig thymocytes were detected in low numbers. However, CCR4(+) T helper cells constituted approximately 9% of the T lymphocyte population within the normal guinea pig lung and 52% of the guinea pig bronchoalveolar lavage fluid, which is consistent with a role for CCR4 in T lymphocyte development and trafficking through normal tissues. Subsequent analysis of chimeric chemokine receptors indicated that 10E4, a functional inhibitor of gpCCR4 responses, recognized the amino terminus of CCR4.