RESUMO
The antidepressant effect of eicosapentaenoic acid-derived bioactive lipid, resolvin E1 (RvE1), was examined in a murine model of chronic pain-induced depression using a tail suspension test. Because RvE1 reportedly possesses agonistic activity on a chemerin receptor ChemR23, we also examined the antidepressant effect of chemerin. Two weeks after surgery for unilateral spared nerve injury to prepare neuropathic pain model mice, immobility time was measured in a tail suspension test. Chronic pain significantly increased immobility time, and this depression-like behavior was attenuated by intracerebroventricular injection of RvE1 (1 ng) or chemerin (500 ng). These results demonstrate that RvE1 exerts an antidepressant effect in a murine model of chronic pain-induced depression, which is likely to be via ChemR23. RvE1 and its receptor may be promising targets to develop novel antidepressants.
Assuntos
Antidepressivos/administração & dosagem , Dor Crônica/complicações , Depressão/tratamento farmacológico , Ácido Eicosapentaenoico/análogos & derivados , Receptores de Quimiocinas/agonistas , Animais , Quimiocinas/administração & dosagem , Quimiocinas/metabolismo , Dor Crônica/psicologia , Depressão/etiologia , Depressão/psicologia , Modelos Animais de Doenças , Ácido Eicosapentaenoico/administração & dosagem , Humanos , Injeções Intraventriculares , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Receptores de Quimiocinas/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
We report the first isolation of the alkaloid aaptamine from the Philippine marine sponge Stylissa sp. Aaptamine possessed weak antiproliferative activity against HCT116 colon cancer cells and inhibited the proteasome in vitro at 50 µM. These activities may be functionally linked. Due to its known, more potent activity on certain G-protein coupled receptors (GPCRs), including α-adrenergic and δ-opioid receptors, the compound was profiled more broadly at sub-growth inhibitory concentrations against a panel of 168 GPCRs to potentially reveal additional targets and therapeutic opportunities. GPCRs represent the largest class of drug targets. The primary screen at 20 µM using the ß-arrestin functional assay identified the antagonist, agonist, and potentiators of agonist activity of aaptamine. Dose-response analysis validated the α-adrenoreceptor antagonist activity of aaptamine (ADRA2C, IC50 11.9 µM) and revealed the even more potent antagonism of the ß-adrenoreceptor (ADRB2, IC50 0.20 µM) and dopamine receptor D4 (DRD4, IC50 6.9 µM). Additionally, aaptamine showed agonist activity on selected chemokine receptors, by itself (CXCR7, EC50 6.2 µM; CCR1, EC50 11.8 µM) or as a potentiator of agonist activity (CXCR3, EC50 31.8 µM; CCR3, EC50 16.2 µM). These GPCRs play a critical role in the treatment of cardiovascular disease, diabetes, cancer, and neurological disorders. The results of this study may thus provide novel preventive and therapeutic strategies for noncommunicable diseases (NCDs).
Assuntos
Alcaloides/farmacologia , Naftiridinas/farmacologia , Doenças não Transmissíveis/tratamento farmacológico , Poríferos/química , Antagonistas Adrenérgicos/farmacologia , Alcaloides/química , Alcaloides/isolamento & purificação , Regulação Alostérica/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Antagonistas de Dopamina/farmacologia , Humanos , Naftiridinas/química , Naftiridinas/isolamento & purificação , Filipinas , Receptores Adrenérgicos/efeitos dos fármacos , Receptores de Quimiocinas/agonistas , Receptores de Quimiocinas/efeitos dos fármacos , Receptores Dopaminérgicos/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
The chemokine-like receptor 1 (CMKLR1) is a promising target for treating autoinflammatory diseases, cancer, and reproductive disorders. However, the interaction between CMKLR1 and its protein-ligand chemerin remains uncharacterized, and no drugs targeting this interaction have passed clinical trials. Here, we identify the binding mode of chemerin-9, the C-terminus of chemerin, at the receptor by combining complementary mutagenesis with structure-based modeling. Incorporating our experimental data, we present a detailed model of this binding site, including experimentally confirmed pairwise interactions for the most critical ligand residues: Chemerin-9 residue F8 binds to a hydrophobic pocket in CMKLR1 formed by the extracellular loop (ECL) 2, while F6 interacts with Y2.68, suggesting a turn-like structure. On the basis of this model, we created the first cyclic peptide with nanomolar activity, confirming the overall binding conformation. This constrained agonist mimics the loop conformation adopted by the natural ligand and can serve as a lead compound for future drug design.
Assuntos
Quimiocinas/química , Quimiocinas/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Receptores de Quimiocinas/agonistas , Animais , Sítios de Ligação , Bovinos , Descoberta de Drogas , Humanos , Camundongos , Simulação de Acoplamento Molecular , Conformação Proteica , Receptores de Quimiocinas/metabolismoRESUMO
Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.
Assuntos
Armadilhas Extracelulares/metabolismo , Neoplasias Experimentais/terapia , Receptores de Quimiocinas/agonistas , Receptores de Interleucina-8A/agonistas , Receptores de Interleucina-8B/agonistas , Animais , Linhagem Celular Tumoral , Citotoxicidade Imunológica/imunologia , Células HT29 , Humanos , Microscopia Intravital/métodos , Células Matadoras Naturais/imunologia , Ligantes , Camundongos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Receptores de Quimiocinas/imunologia , Receptores de Quimiocinas/metabolismo , Receptores de Interleucina-8A/imunologia , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/imunologia , Receptores de Interleucina-8B/metabolismo , Linfócitos T Citotóxicos/imunologiaRESUMO
Chemerin, a chemoattractant protein and adipokine, has been identified as the endogenous ligand for a G protein-coupled receptor encoded by the gene CMKLR1 (also known as ChemR23), and as a consequence the receptor protein was renamed the chemerin receptor in 2013. Since then, chemerin has been identified as the endogenous ligand for a second G protein-coupled receptor, encoded by the gene GPR1 Therefore, the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification recommends that the official name of the receptor protein for chemokine-like receptor 1 (CMKLR1) is chemerin receptor 1, and G protein-coupled receptor 1 is chemerin receptor 2 to follow the convention of naming the receptor protein after the endogenous ligand. Chemerin receptor 1 and chemerin receptor 2 can be abbreviated to Chemerin1 and Chemerin2, respectively. Chemerin requires C-terminal processing for activity, and human chemerin21-157 is reported to be the most active form, with peptide fragments derived from the C terminus biologically active at both receptors. Small-molecule antagonist, CCX832, selectively blocks CMKLR1, and resolvin E1 activation of CMKLR1 is discussed. Activation of both receptors by chemerin is via coupling to Gi/o, causing inhibition of adenylyl cyclase and increased Ca2+ flux. Receptors and ligand are widely expressed in humans, rats, and mice, and both receptors share â¼80% identity across these species. CMKLR1 knockout mice highlight the role of this receptor in inflammation and obesity, and similarly, GPR1 knockout mice exhibit glucose intolerance. In addition, the chemerin receptors have been implicated in cardiovascular disease, cancer, steroidogenesis, human immunodeficiency virus replication, and neurogenerative disease.
Assuntos
Receptores de Quimiocinas/agonistas , Receptores de Quimiocinas/antagonistas & inibidores , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Humanos , Receptores de Quimiocinas/química , Receptores de Quimiocinas/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Terminologia como AssuntoRESUMO
The NLRP3 inflammasome controls interleukin-1ß maturation in antigen-presenting cells, but a direct role for NLRP3 in human adaptive immune cells has not been described. We found that the NLRP3 inflammasome assembles in human CD4(+) T cells and initiates caspase-1-dependent interleukin-1ß secretion, thereby promoting interferon-γ production and T helper 1 (T(H)1) differentiation in an autocrine fashion. NLRP3 assembly requires intracellular C5 activation and stimulation of C5a receptor 1 (C5aR1), which is negatively regulated by surface-expressed C5aR2. Aberrant NLRP3 activity in T cells affects inflammatory responses in human autoinflammatory disease and in mouse models of inflammation and infection. Our results demonstrate that NLRP3 inflammasome activity is not confined to "innate immune cells" but is an integral component of normal adaptive T(H)1 responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Proteínas de Transporte/metabolismo , Complemento C5a/imunologia , Inflamassomos/imunologia , Interferon gama/biossíntese , Células Th1/imunologia , Imunidade Adaptativa , Animais , Comunicação Autócrina , Proteínas de Transporte/genética , Ativação do Complemento , Síndromes Periódicas Associadas à Criopirina/imunologia , Modelos Animais de Doenças , Células HEK293 , Humanos , Imunidade Inata , Inflamação/imunologia , Proteína Cofatora de Membrana/imunologia , Camundongos , Camundongos Mutantes , Proteína 3 que Contém Domínio de Pirina da Família NLR , Espécies Reativas de Oxigênio/metabolismo , Receptor da Anafilatoxina C5a/agonistas , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/metabolismo , Receptores de Antígenos de Linfócitos T/agonistas , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Quimiocinas/agonistas , Receptores de Quimiocinas/antagonistas & inibidores , Receptores de Quimiocinas/metabolismoRESUMO
Chemokine receptors are involved in various pathologies such as inflammatory diseases, cancer, and HIV infection. Small molecule and antibody-based antagonists have been developed to inhibit chemokine-induced receptor activity. Currently two small molecule inhibitors targeting CXCR4 and CCR5 are on the market for stem cell mobilization and the treatment of HIV infection, respectively. Antibody fragments (e.g., nanobodies) targeting chemokine receptors are primarily orthosteric ligands, competing for the chemokine binding site. This is opposed by most small molecules, which act as allosteric modulators and bind to the receptor at a topographically distinct site as compared to chemokines. Allosteric modulators can be distinguished from orthosteric ligands by unique features, such as a saturable effect and probe dependency. For successful drug development, it is essential to determine pharmacological parameters (i.e., affinity, potency, and efficacy) and the mode of action of potential drugs during early stages of research in order to predict the biological effect of chemokine receptor targeting drugs in the clinic. This chapter explains how the pharmacological profile of chemokine receptor targeting ligands can be determined and quantified using binding and functional experiments.
Assuntos
Quimiocinas/metabolismo , Biologia Molecular/métodos , Terapia de Alvo Molecular/métodos , Receptores de Quimiocinas/metabolismo , Regulação Alostérica , Animais , Ligação Competitiva , Linhagem Celular , Quimiotaxia , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Radioisótopos do Iodo , Ligantes , Ligação Proteica , Receptores de Quimiocinas/agonistas , Receptores de Quimiocinas/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , beta-Arrestinas/metabolismoRESUMO
The chemerin receptor (CMKLR1) is a G protein-coupled receptor found on select immune, epithelial, and dorsal root ganglion/spinal cord neuronal cells. CMKLR1 is primarily coupled to the inhibitory G protein, Gαi, and has been shown to modulate the resolution of inflammation and neuropathic pain. CMKLR1 is activated by both lipid and peptide agonists, resolvin E1 and chemerin, respectively. Notably, these ligands have short half-lives. To expedite the development of long acting, stable chemerin analogs as candidate therapeutics, we used membrane-tethered ligand technology. Membrane-tethered ligands are recombinant proteins comprised of an extracellular peptide ligand, a linker sequence, and an anchoring transmembrane domain. Using this technology, we established that a 9-amino acid-tethered chemerin fragment (amino acids 149-157) activates both mouse and human CMKLR1 with efficacy exceeding that of the full-length peptide (amino acids 21-157). To enable in vivo delivery of a corresponding soluble membrane anchored ligand, we generated lipidated analogs of the 9-amino acid fragment. Pharmacological assessment revealed high potency and wash resistance (an index of membrane anchoring). When tested in vivo, a chemerin SMAL decreased allergic airway inflammation and attenuated neuropathic pain in mice. This compound provides a prototype membrane-anchored peptide for the treatment of inflammatory disease. A parallel approach may be applied to developing therapeutics targeting other peptide hormone G protein-coupled receptors.
Assuntos
Asma/tratamento farmacológico , Quimiocinas/farmacologia , Fatores Quimiotáticos/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Neuralgia/tratamento farmacológico , Peptídeos/farmacologia , Receptores de Quimiocinas/agonistas , Receptores Acoplados a Proteínas G/agonistas , Animais , Asma/genética , Asma/metabolismo , Quimiocinas/química , Quimiocinas/genética , Fatores Quimiotáticos/química , Fatores Quimiotáticos/genética , Células HEK293 , Humanos , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos , Neuralgia/genética , Neuralgia/metabolismo , Peptídeos/química , Peptídeos/genética , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
G protein-coupled chemokine receptors and their peptidergic ligands are interesting therapeutic targets due to their involvement in various immune-related diseases, including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, chronic obstructive pulmonary disease, HIV-1 infection and cancer. To tackle these diseases, a lot of effort has been focused on discovery and development of small-molecule chemokine receptor antagonists. This has been rewarded by the market approval of two novel chemokine receptor inhibitors, AMD3100 (CXCR4) and Maraviroc (CCR5) for stem cell mobilization and treatment of HIV-1 infection respectively. The recent GPCR crystal structures together with mutagenesis and pharmacological studies have aided in understanding how small-molecule ligands interact with chemokine receptors. Many of these ligands display behaviour deviating from simple competition and do not interact with the chemokine binding site, providing evidence for an allosteric mode of action. This review aims to give an overview of the evidence supporting modulation of this intriguing receptor family by a range of ligands, including small molecules, peptides and antibodies. Moreover, the computer-assisted modelling of chemokine receptor-ligand interactions is discussed in view of GPCR crystal structures. Finally, the implications of concepts such as functional selectivity and chemokine receptor dimerization are considered.
Assuntos
Receptores de Quimiocinas/metabolismo , Animais , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Quimiocinas/metabolismo , Desenho de Fármacos , Humanos , Receptores de Quimiocinas/agonistas , Receptores de Quimiocinas/antagonistas & inibidoresRESUMO
The highly constitutively active G-protein coupled receptor US28 of human cytomegalovirus (HCMV) is an interesting pharmacological target because of its implication on viral dissemination, cardiovascular diseases and tumorigenesis. We found that dihydroisoquinolinone and tetrahydroisoquinoline scaffolds may be promising lead structures for novel US28 allosteric inverse agonists. These scaffolds were rapidly synthesized by radical carboamination reactions followed by non-radical transformations. Our novel US28 allosteric modulators provide valuable scaffolds for further ligand optimization and may be helpful chemical tools to investigate molecular mechanisms of US28 constitutive signaling and its role in pathogenesis.
Assuntos
Isoquinolinas/farmacologia , Receptores de Quimiocinas/agonistas , Proteínas Virais/agonistas , Regulação Alostérica/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Isoquinolinas/síntese química , Isoquinolinas/química , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Human cytomegalovirus (HCMV) is a widespread human pathogen, possessing onco-modulatory properties. Constitutive signaling of the HCMV-encoded chemokine receptor US28 and its ability to bind a broad spectrum of chemokines might facilitate HCMV-associated tumor progression. Novel nonpeptidergic chemotypes were identified as neutral antagonists or inverse agonists on US28, that allosterically inhibit chemokine binding to US28.
Assuntos
Aminas/farmacologia , Imipramina/farmacologia , Indenos/farmacologia , Receptores de Quimiocinas/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Aminas/síntese química , Aminas/química , Humanos , Imipramina/análogos & derivados , Imipramina/química , Indenos/síntese química , Indenos/química , Ligantes , Estrutura Molecular , Receptores de Quimiocinas/agonistas , Relação Estrutura-Atividade , Proteínas Virais/agonistasRESUMO
Porin of Shigella dysenteriae type 1 up-regulated Toll-like receptor (TLR)2 on CD3-stimulated CD4(+) T cells but could not induce the expression of other TLRs. TLR2 in association with myeloid differentiating factor 88 (MyD88) triggered the downstream signal transduction pathway leading to activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappaB (NF-kappaB), and degradation of IkappaB, the NF-kappaB inhibitor. TLR2 co-stimulation by porin resulted in T cell expansion by inducing both proliferation and survival of the CD4(+) T cells. Extracellular signal-regulated kinase (ERK)1/2 activation inhibitor U0126 and NF-kappaB translocation inhibitor SN-50 significantly inhibited proliferation of T cells, highlighting a direct role of ERK and NF-kappaB in the process. However, cell survival involving Bcl-X(L) induction was found to be regulated essentially by ERK with no significant role of NF-kappaB. Porin-induced proliferation was supported by induction of IL-2 and CD25 that are known to play a pivotal role in T cell expansion. Apart from inducing T cell proliferation, porin triggered effector functions of the cells, evident from TLR2- and MyD88-dependent release of type 1 cytokines tumor necrosis factor (TNF) and interferon (IFN)-gamma along with the induction of type 1 chemokines macrophage-inflammatory protein (MIP)-1alpha and MIP-1beta and their receptor CCR5. The proliferation, survival and effector function of CD4(+) T cells through TLR2 co-stimulation show the capability of porin to directly turn adaptive immunity into action.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Porinas/imunologia , Shigella dysenteriae/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Butadienos/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Citocinas/agonistas , Citocinas/imunologia , Citocinas/metabolismo , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inativação Gênica , Imidazóis/farmacologia , Subunidade alfa de Receptor de Interleucina-2/agonistas , Subunidade alfa de Receptor de Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/imunologia , NF-kappa B/metabolismo , Nitrilas/farmacologia , Peptídeos/farmacologia , Porinas/isolamento & purificação , Porinas/farmacologia , Piridinas/farmacologia , RNA Interferente Pequeno/imunologia , RNA Interferente Pequeno/metabolismo , Receptores de Quimiocinas/agonistas , Receptores de Quimiocinas/imunologia , Receptores de Quimiocinas/metabolismo , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/genéticaRESUMO
A constitutively active G protein-coupled receptor (GPCR) encoded by Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8) (KSHV) is expressed in endothelial (spindle) cells of Kaposi's sarcoma lesions. In this study, we report novel effects of basal signaling by this receptor and of inverse agonist chemokines on migration of KSHV-GPCR-expressing mouse lung endothelial cells. We show that basal signaling by KSHV-GPCR inhibits migration of endothelial cells in two systems, movement through porous filters and in vitro wound closure. Naturally occurring chemokines, interferon gamma-inducible protein-10 and stromal-derived factor-1, which act as inverse agonists at KSHV-GPCR, abrogate the inhibition of migration and stimulate directed migration (or chemotaxis) of these cells. Thus, the expression of KSHV-GPCR may allow infected endothelial cells in situ to remain in a localized environment or to directionally migrate along a gradient of specific chemokines that are inverse agonists at KSHV-GPCR.
Assuntos
Quimiocinas/farmacologia , Quimiotaxia/fisiologia , Agonismo Inverso de Drogas , Células Endoteliais/citologia , Receptores de Quimiocinas/antagonistas & inibidores , Receptores de Quimiocinas/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Quimiocina CXCL1/farmacologia , Quimiocina CXCL10/farmacologia , Quimiocina CXCL11/farmacologia , Quimiocina CXCL12/farmacologia , Quimiocina CXCL9/farmacologia , Quimiotaxia/efeitos dos fármacos , Células Endoteliais/metabolismo , Camundongos , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Receptores de Quimiocinas/agonistas , Transdução de Sinais/efeitos dos fármacos , Transfecção , Cicatrização/efeitos dos fármacos , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Human herpesvirus-6A (HHV-6A) betachemokine-receptor U51A binds inflammatory modulators CCL2, CCL5, CCL11, CCL7, and CCL13. This unique specificity overlaps that of human chemokine receptors CCR1, CCR2, CCR3, and CCR5. In model cell lines, expression leads to CCL5 down-regulation with both constitutive and inducible signaling. Here, immunomodulation pathways are investigated in human leukocytes permissive for infection. Constitutive signaling was shown using inositol phosphate assays and inducible calcium signaling by response to CCL2, CCL5 and CCL11. Constitutive signaling targets were examined using an immune response-related microarray and RT-PCR, showing down-regulation of CCL5 and FOG-2, a hematopoietic transcriptional repressor. By RT-PCR and siRNA reversion, CCL5 and FOG-2 were shown down-regulated, during peak U51A expression post infection. Two further active ligands, XCL1 and CCL19, were identified, making U51A competitor to their human receptors, XCR1 and CCR7, on T lymphocytes, NK and dendritic cells. Finally, U51A-expressing cell lines and infected ex vivo leukocytes, showed migration towards chemokine-gradients, and chemokine internalization. Consequently, U51A may affect virus dissemination or host transmission by chemotaxis of infected cells to sites of chemokine secretion specific for U51A (for example the lymph node or lung, by CCL19 or CCL11, respectively) and evade immune-effector cells by chemokine diversion and down-regulation, affecting virus spread and inflammatory pathology.
Assuntos
Quimiocina CCL5/genética , Proteínas de Ligação a DNA/genética , Leucócitos Mononucleares/metabolismo , Receptores CCR7/genética , Receptores de Quimiocinas/fisiologia , Receptores Acoplados a Proteínas G/genética , Receptores Virais/fisiologia , Fatores de Transcrição/genética , Ligação Competitiva , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Tumoral , Quimiocina CCL11/farmacologia , Quimiocina CCL19/metabolismo , Quimiocina CCL19/farmacologia , Quimiocina CCL2/metabolismo , Quimiocina CCL2/farmacologia , Quimiocina CCL5/farmacologia , Quimiocinas/metabolismo , Quimiocinas/farmacologia , Quimiocinas C/metabolismo , Quimiocinas C/farmacologia , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/imunologia , Regulação para Baixo/genética , Endocitose/efeitos dos fármacos , Endocitose/imunologia , Herpesvirus Humano 6/imunologia , Humanos , Fosfatos de Inositol/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Mimetismo Molecular , RNA Interferente Pequeno/genética , Receptores de Quimiocinas/agonistas , Receptores Virais/agonistasRESUMO
Stem cell research is currently focused on totipotent stem cells and their therapeutic potential, however adult stem cells, while restricted to differentiation within their tissue or origin, also have therapeutic utility. Transplantation with bone marrow hematopoietic stem cells (HSC) has been used for curative therapy for decades. More recently, alternative sources of HSC, particularly those induced to exit marrow or mobilize to peripheral blood by G-CSF, have become the most widely used hematopoietic graft and show significant superiority to marrow HSC. The chemokine/chemokine receptor axis also mobilizes HSC that occurs more rapidly than with G-CSF. In mice, the HSC and progenitor cells (HPC) mobilized by the CXCR2 receptor agonist GRObeta can be harvested within minutes of administration and show significantly lower levels of apoptosis, enhanced homing to marrow, expression of more activated integrin receptors and superior repopulation kinetics and more competitive engraftment than the equivalent cells mobilized by G-CSF. These characteristics suggest that chemokine axis-mobilized HSC represent a population of adult stem cells distinct from those mobilized by G-CSF, with superior therapeutic potential. It remains to be determined if the chemokine mobilization axis can be harnessed to mobilize other populations of unique adult stem cells with clinical utility.
Assuntos
Células-Tronco Adultas/efeitos dos fármacos , Quimiocinas/farmacologia , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Receptores de Quimiocinas/agonistas , Células-Tronco Adultas/classificação , Células-Tronco Adultas/citologia , Animais , Quimiocina CXCL12/fisiologia , Quimiocina CXCL2/farmacologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/classificação , Células-Tronco Hematopoéticas/citologia , Humanos , Imunofenotipagem , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Transplante de Células-Tronco de Sangue Periférico , Quimera por Radiação , Receptores de Quimiocinas/fisiologia , Receptores de Interleucina-8B/agonistas , Receptores de Interleucina-8B/efeitos dos fármacosRESUMO
Chemokine receptor CCR3 is highly expressed by eosinophils and signals in response to binding of the eotaxin family of chemokines, which are up-regulated in allergic disorders. Consequently, CCR3 blockade is of interest as a possible therapeutic approach for the treatment of allergic disease. We have described previously a bispecific antagonist of CCR1 and CCR3 named UCB35625 that was proposed to interact with the transmembrane residues Tyr-41, Tyr-113, and Glu-287 of CCR1, all of which are conserved in CCR3. Here, we show that cells expressing the CCR3 constructs Y113A and E287Q are insensitive to antagonism by UCB35625 and also exhibit impaired chemotaxis in response to CCL11/eotaxin, suggesting that these residues are important for antagonist binding and also receptor activation. Furthermore, mutation of the residue Tyr-113 to alanine was found to turn the antagonist UCB35625 into a CCR3 agonist. Screens of small molecule libraries identified a novel specific agonist of CCR3 named CH0076989. This was able to activate eosinophils and transfectants expressing both wild-type CCR3 and a CCR1-CCR3 chimeric receptor lacking the CCR3 amino terminus, indicating that this region of CCR3 is not required for CH0076989 binding. A direct interaction with the transmembrane helices of CCR3 was supported by mutation of the residues Tyr-41, Tyr-113, and Glu-287 that resulted in complete loss of CH0076989 activity, suggesting that the compound mimics activation by CCL11. We conclude that both agonists and antagonists of CCR3 appear to occupy overlapping sites within the transmembrane helical bundle, suggesting a fine line between agonism and antagonism of chemokine receptors.
Assuntos
Receptores de Quimiocinas/agonistas , Receptores de Quimiocinas/antagonistas & inibidores , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Quimiocina CCL11 , Quimiocinas/metabolismo , Quimiocinas CC/química , Regulação para Baixo , Eosinófilos/metabolismo , Ácido Glutâmico/química , Camundongos , Modelos Biológicos , Conformação Molecular , Receptores CCR3 , Receptores de Quimiocinas/fisiologia , Tirosina/química , Regulação para Cima , Xantenos/farmacologiaRESUMO
The granulomatous and intramural inflammation observed in cases of inflammatory bowel diseases (IBD) and veterinary Johne's disease suggests that Mycobacterium avium subsp. paratuberculosis is a causative agent. However, an incomplete understanding of the immunological steps responsible for the pathologies of IBD makes this conclusion uncertain. Sera from interleukin-10-deficient (IL-10(-/-)) mice with spontaneous colitis displayed significantly higher M. avium subsp. paratuberculosis-specific immunoglobulin G2a antibody responses than did sera from similar mice without disease. Pathogen-free IL-10(-/-) mice received control vehicle or the vehicle containing heat-killed or live M. avium subsp. paratuberculosis. Mucosal CD4(+) T cells from the mice that developed colitis proliferated and secreted higher levels of gamma interferon and tumor necrosis factor alpha after ex vivo stimulation with a Vbeta11(+) T-cell receptor-restricted peptide from the MPT59 antigen (Ag85B) than those secreted from cells from mice before the onset of colitis. The data from this study provide important information regarding the mechanisms of colitis in IL-10(-/-) mice, which are driven in part by Ag85B-specific T cells. The data suggest a plausible mechanism of Ag-specific T-cell responses in colitis driven by potent Ags conserved in Mycobacterium species.
Assuntos
Colite/microbiologia , Colite/patologia , Mycobacterium avium subsp. paratuberculosis/imunologia , Paratuberculose/imunologia , Paratuberculose/patologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Colite/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Interferon gama/biossíntese , Interleucina-10/deficiência , Mucosa Intestinal/imunologia , Ligantes , Camundongos , Camundongos Knockout , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores CXCR3 , Receptores de Quimiocinas/agonistas , Receptores de Quimiocinas/imunologia , Organismos Livres de Patógenos Específicos , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Células Th1/imunologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Resolvin E1 (RvE1) is a potent anti-inflammatory and proresolving mediator derived from omega-3 eicosapentaenoic acid generated during the resolution phase of inflammation. RvE1 possesses a unique structure and counterregulatory actions that stop human polymorphonuclear leukocyte (PMN) transendothelial migration and PMN infiltration in several murine inflammatory models. To examine the mechanism(s) underlying anti-inflammatory actions on PMNs, we prepared [(3)H]RvE1 and characterized its interactions with human PMN. Results with membrane fractions of human PMN demonstrated specific binding with a K(d) of 48.3 nM. [(3)H]RvE1 specific binding to human PMN was displaced by leukotriene B(4) (LTB(4)) and LTB(4) receptor 1 (BLT1) antagonist U-75302, but not by chemerin peptide, a ligand specific for another RvE1 receptor ChemR23. Recombinant human BLT1 gave specific binding with [(3)H]RvE1 with a K(d) of 45 nM. RvE1 selectively inhibited adenylate cyclase with BLT1, but not with BLT2. In human PBMC, RvE1 partially induced calcium mobilization, and blocked subsequent stimulation by LTB(4). RvE1 also attenuated LTB(4)-induced NF-kappaB activation in BLT1-transfected cells. In vivo anti-inflammatory actions of RvE1 were sharply reduced in BLT1 knockout mice when given at low doses (100 ng i.v.) in peritonitis. In contrast, RvE1 at higher doses (1.0 mug i.v.) significantly reduced PMN infiltration in a BLT1-independent manner. These results indicate that RvE1 binds to BLT1 as a partial agonist, potentially serving as a local damper of BLT1 signals on leukocytes along with other receptors (e.g., ChemR23-mediated counterregulatory actions) to mediate the resolution of inflammation.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Ácido Eicosapentaenoico/análogos & derivados , Neutrófilos/metabolismo , Receptores de Quimiocinas/agonistas , Receptores do Leucotrieno B4/agonistas , Animais , Sinalização do Cálcio/fisiologia , Movimento Celular/fisiologia , Quimiocinas/farmacologia , Relação Dose-Resposta a Droga , Ácido Eicosapentaenoico/farmacologia , Álcoois Graxos/farmacologia , Glicóis/farmacologia , Humanos , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Ligantes , Camundongos , Camundongos Knockout , Neutrófilos/citologia , Peptídeos/farmacologia , Receptores de Quimiocinas/antagonistas & inibidores , Receptores de Quimiocinas/metabolismo , Receptores do Leucotrieno B4/antagonistas & inibidores , Receptores do Leucotrieno B4/deficiência , Receptores do Leucotrieno B4/metabolismoRESUMO
Expression of CXCR3-targeting chemokines have been demonstrated in several diseases, suggesting a critical role for CXCR3 in recruiting activated T cells to sites of immune-mediated inflammation. Sjögren's syndrome (SS) is an autoimmune disease characterized by a mononuclear cell infiltrate of activated T cells around the duct in the salivary gland. Analysis of minor salivary gland biopsy specimens from 20 healthy subjects and 18 patients with primary SS demonstrated that CXCR3, in particular, the B form of this receptor, is constitutively expressed by human salivary gland epithelial cells. Salivary gland epithelial cell cultures demonstrated that CXCR3 participate in removing relevant amount of agonists from the supernatant of exposed cells without mediating calcium flux or chemotaxis while retaining the ability to undergo internalization. Although in normal salivary gland epithelial cells, CXCR3 behaves as a chemokine-scavenging receptor, its role in SS cells is functionally impaired. The impairment of this scavenging function might favor chemotaxis, leading to heightened immigration of CXCR3-positive T lymphocytes. These findings suggest that epithelial CXCR3 may be involved in postsecretion regulation of chemokine bioavailability. They also support a critical role for CXCR3 in the pathogenesis of SS and identify its agonists as potential therapeutic targets.
Assuntos
Quimiocinas/metabolismo , Células Epiteliais/metabolismo , Receptores de Quimiocinas/metabolismo , Glândulas Salivares/metabolismo , Síndrome de Sjogren/metabolismo , Adulto , Idoso , Disponibilidade Biológica , Células Cultivadas , Quimiocina CXCL10 , Quimiocinas CXC/metabolismo , Quimiotaxia , Células Epiteliais/citologia , Feminino , Humanos , Ligantes , Pessoa de Meia-Idade , Receptores CXCR3 , Receptores de Quimiocinas/agonistas , Glândulas Salivares/citologia , Glândulas Salivares/imunologia , Síndrome de Sjogren/imunologiaRESUMO
We have earlier shown that the rat neural progenitor cell line HiB5 is capable of suppressing intracranial growth of glioma cells in Fisher rats. Unlike some neural progenitor cells, HiB5 cells have not shown homing capacity towards glioma cells growing intracranially. In this study, we have genetically modified HiB5 progenitor cells to over-express the chemokine receptor CXCR3. We show that the introduced receptor is functionally responding to ligand stimulation with increased phosphorylation levels of ERK and SAPK/JNK and a transcriptional response of an AP-1 reporter system introduced into HIB5 cells. These transfected progenitor cells migrate in vitro in response to IP-10 and I-TAC. Further, we show an enhanced in vivo migration of the CXCR3 transfected HiB5 cells over the corpus callosum towards an IP-10 and I-TAC expressing glioma, as compared to wild type HiB5 cells. Our data indicate that it is possible to take advantage of chemokines natural capacity to initiate migratory responses, and to use this ability to enhance tumor-inhibitory neural progenitor cells to target an intracranially growing glioma.