RESUMO
During inflammatory response, blood leukocytes adhere to the endothelium. This process involves numerous adhesion molecules, including a transmembrane chemokine, CX3CL1, which behaves as a molecular cluster. How this cluster assembles and whether this association has a functional role remain unknown. The analysis of CX3CL1 clusters using native electrophoresis and single molecule fluorescence kinetics shows that CX3CL1 is a homo-oligomer of 3 to 7 monomers. Fluorescence recovery after photobleaching assays reveal that the CX3CL1-transmembrane domain peptide self-associates in both cellular and acellular lipid environments, while its random counterpart (i.e. peptide with the same residues in a different order) does not. This strongly indicates that CX3CL1 oligomerization is driven by its intrinsic properties. According to the molecular modeling, CX3CL1 does not associate in compact bundles but rather with monomers linearly assembled side by side. Finally, the CX3CL1 transmembrane peptide inhibits both the CX3CL1 oligomerization and the adhesive function, while its random counterpart does not. This demonstrates that CX3CL1 oligomerization is mandatory for its adhesive potency. Our results provide a new direction to control CX3CL1-dependent cellular adherence in key immune processes.
Assuntos
Adesão Celular/fisiologia , Quimiocina CX3CL1/metabolismo , Animais , Células CHO , Células COS , Linhagem Celular , Chlorocebus aethiops , Cricetulus , Células HEK293 , Humanos , Proteínas de Membrana/metabolismoRESUMO
Sepsis is characterized by a systemic inflammation that can cause an immune dysfunction, for which the underlying mechanisms are unclear. We investigated the impact of cecal ligature and puncture (CLP)-mediated polymicrobial sepsis on monocyte (Mo) mobilization and functions. Our results show that CLP led to two consecutive phases of Mo deployment. The first one occurred within the first 3 days after the induction of the peritonitis, while the second phase was of a larger amplitude and extended up to a month after apparent clinical recovery. The latter was associated with the expansion of Mo in the tissue reservoirs (bone marrow and spleen), their release in the blood and their accumulation in the vasculature of peripheral non-lymphoid tissues. It occurred even after antibiotic treatment but relied on inflammatory-dependent pathways and inversely correlated with increased susceptibility and severity to a secondary infection. The intravascular lung Mo displayed limited activation capacity, impaired phagocytic functions and failed to transfer efficient protection against a secondary infection into monocytopenic CCR2-deficient mice. In conclusion, our work unveiled key dysfunctions of intravascular inflammatory Mo during the recovery phase of sepsis and provided new insights to improve patient protection against secondary infections.
Assuntos
Inflamação/imunologia , Monócitos/imunologia , Sepse/imunologia , Animais , Antígenos Ly/análise , Receptor 1 de Quimiocina CX3C/fisiologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/fisiologia , Neutrófilos/imunologia , Fagocitose , Receptores CCR2/fisiologiaRESUMO
The recent decline in global malaria burden has stimulated efforts toward Plasmodium falciparum elimination. Understanding the biology of malaria transmission stages may provide opportunities to reduce or prevent onward transmission to mosquitoes. Immature P. falciparum transmission stages, termed stages I to IV gametocytes, sequester in human bone marrow before release into the circulation as mature stage V gametocytes. This process likely involves interactions between host receptors and potentially immunogenic adhesins on the infected red blood cell (iRBC) surface. Here, we developed a flow cytometry assay to examine immune recognition of live gametocytes of different developmental stages by naturally exposed Malawians. We identified strong antibody recognition of the earliest immature gametocyte-iRBCs (giRBCs) but not mature stage V giRBCs. Candidate surface antigens (n = 30), most of them shared between asexual- and gametocyte-iRBCs, were identified by mass spectrometry and mouse immunizations, as well as correlations between responses by protein microarray and flow cytometry. Naturally acquired responses to a subset of candidate antigens were associated with reduced asexual and gametocyte density, and plasma samples from malaria-infected individuals were able to induce immune clearance of giRBCs in vitro. Infected RBC surface expression of select candidate antigens was validated using specific antibodies, and genetic analysis revealed a subset with minimal variation across strains. Our data demonstrate that humoral immune responses to immature giRBCs and shared iRBC antigens are naturally acquired after malaria exposure. These humoral immune responses may have consequences for malaria transmission potential by clearing developing gametocytes, which could be leveraged for malaria intervention.
Assuntos
Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Plasmodium falciparum/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Anticorpos Antiprotozoários/metabolismo , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Eritrócitos/parasitologia , Citometria de Fluxo , Humanos , Immunoblotting , Malária/imunologia , Malária/metabolismo , Malária/prevenção & controle , Malária Falciparum/prevenção & controle , Camundongos , Microscopia de Fluorescência , Fagocitose/fisiologia , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Espectrometria de Massas em TandemRESUMO
Malaria caused by Plasmodium falciparum is associated with cytoadherence of infected red blood cells (iRBC) to endothelial cells. Numerous host molecules have been involved in cytoadherence, including the adhesive chemokine CX3CL1. Most of the identified parasite ligands are from the multigenic and hypervariable Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family which makes them poor targets for the development of a broadly protective vaccine. Using proteomics, we have identified two 25-kDa parasite proteins with adhesive properties for CX3CL1, called CBP for CX3CL1 Binding Proteins. CBPs are coded by single-copy genes with little polymorphic variation and no homology with other P. falciparum gene products. Specific antibodies raised against epitopes from the predicted extracellular domains of each CBP efficiently stain the surface of RBC infected with trophozoites or schizonts, which is a strong indication of CBP expression at the surface of iRBC. These anti-CBP antibodies partially neutralize iRBC adherence to CX3CL1. This adherence is similarly inhibited in the presence of peptides from the CBP extracellular domains, while irrelevant peptides had no such effect. CBP1 and CBP2 are new P. falciparum ligands for the human chemokine CX3CL1. The identification of this non-polymorphic P. falciparum factors provides a new avenue for innovative vaccination approaches.
RESUMO
CC chemokine receptor type 2 (CCR2) is a key molecule in inflammatory diseases and is an obvious drug target for the treatment of inflammation. A number of nonpeptidic, competitive CCR2 antagonists have been developed, but none has yet been approved for clinical use. Our aim was to identify a short peptide that showed allosteric antagonism against human and mouse CCR2. On the basis of sequence analysis and 3-dimensional modeling, we identified an original 7-d-amino acid peptidic CCR2 inhibitor that we have called extracellular loop 1 inverso (ECL1i), d(LGTFLKC). In vitro, ECL1i selectively and potently inhibits CC chemokine ligand type 2 (CCL2)-triggered chemotaxis (IC50, 2 µM) but no other conventional CCL2-associated events. We used the classic competitive CCR2 antagonist, BMS22 {2-[(isopropylaminocarbonyl)amino]-N-[2-[[cis-2-[[4-(methylthio)benzoyl]amino]cyclohexyl]amino]-2-oxoethyl]-5-(trifluoromethyl)benzamide}, as positive control and inhibited CCL2-dependent chemotaxis with an IC50 of 18 nM. As negative control, we used a peptide with the same composition as ECL1i, but in a different sequence, d(FKLTLCG). In vivo, ECL1i (4 mg/kg) interfered with CCR2-positive cell recruitment and attenuated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. This study establishes ECL1i as the first allosteric inhibitor of CCR2 with functional selectivity. ECL1i is a promising new agent in therapeutic development, and it may, by its selective effect, increase our understanding of CCR2 signaling pathways and functions.-Auvynet, C., Baudesson de Chanville, C., Hermand, P., Dorgham, K., Piesse, C., Pouchy, C., Carlier, L., Poupel, L., Barthélémy, S., Felouzis, V., Lacombe, C., Sagan, S., Salomon, B., Deterre, P., Sennlaub, F., Combadière, C. ECL1i, d(LGTFLKC), a novel, small peptide that specifically inhibits CCL2-dependent migration.
Assuntos
Movimento Celular/fisiologia , Quimiocina CCL2/metabolismo , Oligopeptídeos/farmacologia , Receptores CCR2/metabolismo , Animais , Células CHO , Quimiocina CCL2/genética , Cricetulus , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Modelos Moleculares , Oligopeptídeos/química , Ligação Proteica , Conformação Proteica , Receptores CCR2/genéticaRESUMO
Monocytes have a crucial role in both proinflammatory and anti-inflammatory phenomena occurring during sepsis. Monocyte recruitment and activation are orchestrated by the chemokine receptors CX3CR1 and CCR2 and their cognate ligands. However, little is known about the roles of these cells and chemokines during the acute phase of inflammation in sepsis. Using intravital microscopy in a murine model of polymicrobial sepsis, we showed that inflammatory Ly6C(high) monocytes infiltrated kidneys, exhibited altered motility, and adhered strongly to the renal vascular wall in a chemokine receptor CX3CR1-dependent manner. Adoptive transfer of Cx3cr1-proficient monocyte-enriched bone marrow cells into septic Cx3cr1-depleted mice prevented kidney damage and promoted mouse survival. Modulation of CX3CR1 activation in septic mice controlled monocyte adhesion, regulated proinflammatory and anti-inflammatory cytokine expression, and was associated with the extent of kidney lesions such that the number of lesions decreased when CX3CR1 activity increased. Consistent with these results, the pro-adhesive I249 CX3CR1 allele in humans was associated with a lower incidence of AKI in patients with sepsis. These data show that inflammatory monocytes have a protective effect during sepsis via a CX3CR1-dependent adhesion mechanism. This receptor might be a new therapeutic target for kidney injury during sepsis.
Assuntos
Injúria Renal Aguda/prevenção & controle , Reação de Fase Aguda/imunologia , Adesão Celular , Monócitos/transplante , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Sepse/complicações , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/patologia , Transferência Adotiva , Alelos , Animais , Antígenos Ly/análise , Receptor 1 de Quimiocina CX3C , Adesão Celular/genética , Movimento Celular , Endotélio Vascular/metabolismo , Genótipo , Humanos , Microscopia Intravital , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência por Excitação Multifotônica , Monócitos/química , Monócitos/fisiologia , Polimorfismo Genético , Receptores de Interleucina-1/antagonistas & inibidoresRESUMO
Chemokine receptors are members of the G-protein-coupled receptor (GPCR) family coupled to members of the Gi class, whose primary function is to inhibit the cellular adenylate cyclase. We used a cAMP-related and PKA-based luminescent biosensor (GloSensor™ F-22) to monitor the real-time downstream response of chemokine receptors, especially CX3CR1 and CXCR4, after activation with their cognate ligands CX3CL1 and CXCL12. We found that the amplitudes and kinetic profiles of the chemokine responses were conserved in various cell types and were independent of the nature and concentration of the molecules used for cAMP prestimulation, including either the adenylate cyclase activator forskolin or ligands mediating Gs-mediated responses like prostaglandin E2 or beta-adrenergic agonist. We conclude that the cAMP chemokine response is robustly conserved in various inflammatory conditions. Moreover, the cAMP-related luminescent biosensor appears as a valuable tool to analyze the details of Gi-mediated cAMP-inhibitory cellular responses, even in native conditions and could help to decipher their precise role in cell function.
Assuntos
Adenilil Ciclases/metabolismo , Técnicas Biossensoriais , Quimiocinas/metabolismo , Colforsina/farmacologia , AMP Cíclico/metabolismo , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Isoproterenol/farmacologia , Cinética , Receptores CXCR4/efeitos dos fármacosRESUMO
The multi-domain CX3CL1 transmembrane chemokine triggers leukocyte adherence without rolling and migration by presenting its chemokine domain (CD) to its receptor CX3CR1. Through the combination of functional adhesion assays with structural analysis using FRAP, we investigated the functional role of the other domains of CX3CL1, i.e., its mucin stalk, transmembrane domain, and cytosolic domain. Our results indicate that the CX3CL1 molecular structure is finely adapted to capture CX3CR1 in circulating cells and that each domain has a specific purpose: the mucin stalk is stiffened by its high glycosylation to present the CD away from the membrane, the transmembrane domain generates the permanent aggregation of an adequate amount of monomers to guarantee adhesion and prevent rolling, and the cytosolic domain ensures adhesive robustness by interacting with the cytoskeleton. We propose a model in which quasi-immobile CX3CL1 bundles are organized to quickly generate adhesive patches with sufficiently high strength to capture CX3CR1+ leukocytes but with sufficiently low strength to allow their patrolling behavior.
RESUMO
OBJECTIVE: Alterations of the chemokine receptor CX3CR1 gene were associated with a reduced risk of myocardial infarction in human and limited atherosclerosis in mice. In this study, we addressed whether CX3CR1 antagonists are potential therapeutic tools to limit acute and chronic inflammatory processes in atherosclerosis. APPROACH AND RESULTS: Treatment with F1, an amino terminus-modified CX3CR1 ligand endowed with CX3CR1 antagonist activity, reduced the extent of atherosclerotic lesions in both Apoe(-/-) and Ldlr(-/-) proatherogenic mouse models. Macrophage accumulation in the aortic sinus was reduced in F1-treated Apoe(-/-) mice but the macrophage density of the lesions was similar in F1-treated and control mice. Both in vitro and in vivo F1 treatment reduced CX3CR1-dependent inflammatory monocyte adhesion, potentially limiting their recruitment. In addition, F1-treated Apoe(-/-) mice displayed reduced numbers of blood inflammatory monocytes, whereas resident monocyte numbers remained unchanged. Both in vitro and in vivo F1 treatment reduced CX3CR1-dependent inflammatory monocyte survival. Finally, F1 treatment of Apoe(-/-) mice with advanced atherosclerosis led to smaller lesions than untreated mice but without reverting to the initial phenotype. CONCLUSIONS: The CX3CR1 antagonist F1 is a potent inhibitor of the progression of atherosclerotic lesions by means of its selective impact on inflammatory monocyte functions. Controlling monocyte trafficking and survival may be an alternative or complementary therapy to lipid-lowering drugs classically used in the treatment of atherosclerosis.
Assuntos
Anti-Inflamatórios/farmacologia , Aorta/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Quimiocina CX3CL1/farmacologia , Hipolipemiantes/farmacologia , Peptídeos/farmacologia , Receptores de Quimiocinas/antagonistas & inibidores , Animais , Aorta/imunologia , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/imunologia , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Receptor 1 de Quimiocina CX3C , Células Cultivadas , Modelos Animais de Doenças , Humanos , Ligantes , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Receptores de Quimiocinas/metabolismo , Receptores de LDL/deficiência , Receptores de LDL/genética , Fatores de TempoRESUMO
The chemokine receptor CCR2 controls the release of Ly6C(high) monocytes from the bone marrow and their recruitment to sites of inflammation. A second chemokine receptor, CX3CR1, is differentially expressed on monocyte subsets. We examined the role of CX3CR1 in monocyte trafficking during the recovery phase after cyclophosphamide (CP)-induced myeloablation and observed that, in the absence of CCR2, Ly6C(high) monocytes accumulated in the bone marrow and peripheral reconstitution was severely impaired compared with wild-type (WT) mice. In contrast, in the absence of CX3CR1, Ly6C(high) monocytes accumulated less rapidly in the marrow but recovered faster in the blood and were more recruited into the spleen, suggesting an opposite action between CCR2 and CX3CR1 in myelorestoration. During the recovery phase, marrow medullar monocytes displayed lower CX3CR1 expression and reduced their adherence to coated CX3CL1. Intravital imaging of the bone marrow showed that CP treatment impacts monocyte trafficking between the parenchyma and the vasculature. Medullar monocytes in CX3CR1(-/-) mice and mice treated with a specific antagonist of CX3CR1 displayed increased mean velocity and displacement and a reduced arrest coefficient compared with WT mice. This study indicates that CX3CR1 reduces the motility of Ly6C(high) monocytes in the bone marrow and thereby controls their release.
Assuntos
Antineoplásicos/farmacologia , Medula Óssea , Quimiotaxia de Leucócito/genética , Monócitos/fisiologia , Receptores de Quimiocinas/fisiologia , Animais , Antígenos Ly/metabolismo , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Medula Óssea/fisiologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Receptor 1 de Quimiocina CX3C , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismoRESUMO
Monocyte/macrophages are implicated in initiation of angiogenesis, tissue/organ perfusion and atherosclerosis biology. We recently showed that chemokine receptor CX(3)CR1 is an essential regulator of monocyte/macrophage derived smooth muscle cell differentiation in the vessel wall after injury. Here we hypothesised the contribution of CX(3)CR1- CX(3)CL1 interaction to in vivo neovascularization and studied the functional consequences of genetic and pharmacologic targeting of CX(3)CR1 in formation, maturation and maintenance of microvascular integrity. Cells functionally deficient in CX(3)CR1 lacked matrix tunnelling and tubulation capacity in a 3D Matrigel assay. These morphogenic and cytokinetic responses were driven by CX(3)CL1-CX(3)CR1 interaction and totally abrogated by a Rho antagonist. To evaluate the role of CX(3)CR1 system in vivo, Matrigel plugs were implanted in competent CX(3)CR1(+/gfp) and functionally deficient CX(3)CR1(gfp/gfp) mice. Leaky microvessels (MV) were formed in the Matrigel implanted in CX(3)CR1(gfp/gfp) but not in CX(3)CR1(+/gfp) mice. In experimental plaque neovascularization immature MV phenotype was observed in CX(3)CR1(gfp/gfp) mice, lacking CX(3)CR1 positive smooth muscle-like cells, extracellular collagen and basement membrane (BM) laminin compared to competent CX(3)CR1(+/gfp) mice. This was associated with increased extravasation of platelets into the intima of CX(3)CR1(gfp/gfp) but not functionally competent CX(3)CR1 mice. Pharmacologic targeting using CX(3)CR1 receptor antagonist in wild type mice resulted in formation of plaque MV with poor BM coverage and a leaky phenotype. Our data indicate a hitherto unrecognised role for functional CX(3)CR1 in Matrigel and experimental plaque neovascularization in vivo, which may buttress MV collectively in favour of a more stable non-leaky phenotype.
Assuntos
Endotélio Vascular/metabolismo , Macrófagos/metabolismo , Microvasos/metabolismo , Monócitos/metabolismo , Receptores de Quimiocinas/genética , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/patologia , Receptor 1 de Quimiocina CX3C , Permeabilidade Capilar , Movimento Celular/efeitos dos fármacos , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/metabolismo , Colágeno/genética , Colágeno/metabolismo , Combinação de Medicamentos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Proteínas de Fluorescência Verde , Laminina/genética , Laminina/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microvasos/efeitos dos fármacos , Microvasos/patologia , Monócitos/efeitos dos fármacos , Monócitos/patologia , Neovascularização Patológica , Peptídeos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteoglicanas , Receptores de Quimiocinas/antagonistas & inibidores , Receptores de Quimiocinas/metabolismoRESUMO
The chemokine CX3CL1 is expressed as a membrane protein that forms a potent adhesive pair with its unique receptor CX3CR1. This receptor has 3 natural variants, V249-T280 (VT), I249-T280 (IT), and I249-M280 (IM), whose relative frequencies are significantly associated with the incidence of various inflammatory diseases. To assess the adhesive potency of CX3CR1 and the molecular diversity of its variants, we assayed their clustering status and their possible structural differences by fluorescence/bioluminescence resonance energy transfer (FRET or BRET) techniques. FRET assays by flow cytometry showed that the CX3CR1 variants cluster, in comparison with appropriate controls. BRET assays showed low nonspecific signals for VT and IT variants and high specific signals for IM, and thus pointed out a structural difference in this variant. We used molecular modeling to show how natural point mutations of CX3CR1 affect the packing of the 6th and 7th helices of this G-protein coupled receptor. Moreover, we found that the BRET technique is sensitive enough to detect these tiny changes. Consistently with our previous finding that CX3CL1 aggregates, our data here indicate that CX3CR1 clustering may contribute to the adhesiveness of the CX3CL1-CX3CR1 pair and may thus represent a new target for anti-inflammatory therapies.
Assuntos
Quimiocina CX3CL1/química , Quimiocina CX3CL1/genética , Transferência Ressonante de Energia de Fluorescência , Variação Genética , Modelos Moleculares , Linhagem Celular , Citometria de Fluxo , Humanos , Polimorfismo Genético , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estrutura Terciária de Proteína , Reprodutibilidade dos TestesRESUMO
Interactions between P-sel and the PSGL-1 mediate the earliest adhesive events during an inflammatory response. Human PSGL-1 displays a high degree of genetic polymorphism that has been diversely associated with susceptibility to human diseases. In the central part of PSGL-1, a 10-aa motif is repeated 14, 15, or 16 times. Moreover, two mutations, M62I and M274V, are often found giving the most common variant M62-M274 with 16 motifs (M16M) and its variants I62-M274 (I16M). Two other variants exist with 15 repeated motifs (M62-M274; M15M) and with 14 motifs (M62-V274; M14V). We investigated the potential difference in the adhesive properties between these natural variants stably expressed in the HEK cell line by using the BFP technique. Their interactions with P-sel were found to be of catch bond-type, and the dissociation force was primarily dependent on the number of decameric motifs: the shorter the PSGL-1, the larger the bond strength. Finally, we found that the M62I mutation, which is close to the binding site to P-sel, reduced the adhesiveness to P-sel effectively. Collectively, these data shed new light on the polymorphism of PSGL-1 and could help the research on its associations to human pathologies.
Assuntos
Glicoproteínas de Membrana/metabolismo , Selectina-P/metabolismo , Polimorfismo Genético , Motivos de Aminoácidos , Adesão Celular/fisiologia , Linhagem Celular , Humanos , Glicoproteínas de Membrana/genética , Selectina-P/genética , Ligação ProteicaRESUMO
Chemokines are mainly involved in the recruitment of leukocytes into tissues, a key feature of inflammation. Through its unique receptor CX3CR1, the chemokine CX3CL1 participates in diverse inflammatory processes including arterial atherosclerosis and cerebral or renal inflammation. Using a phage display strategy, we engineered a hCX3CL1 analog (named F1) with a modified N terminus. F1 bound specifically to cells expressing hCX3CR1 and had a K(d) value close to that of native CX3CL1. F1 was not a signaling molecule and did not induce chemotaxis, calcium flux, or CX3CR1 internalization. However, it potently inhibited the CX3CL1-induced calcium flux and chemotaxis in CX3CR1-expressing primary cells of human and murine origin with an IC(50) of 5-50 nM. It also efficiently inhibited the cell adhesion mediated by the CX3CL1-CX3CR1 axis. Finally, in a noninfectious murine model of peritonitis, F1 strongly inhibited macrophage accumulation. These data reveal a prototype molecule that is the first bona fide antagonist of hCX3CR1. This molecule could be used as a lead compound for the development of a novel class of anti-inflammatory substances that act by inhibiting CX3CR1.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Quimiotaxia/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Peptídeos/farmacologia , Receptores de Quimiocinas/antagonistas & inibidores , Animais , Células CHO , Receptor 1 de Quimiocina CX3C , Cálcio/metabolismo , Adesão Celular/efeitos dos fármacos , Quimiocina CX3CL1/metabolismo , Cricetinae , Cricetulus , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Camundongos , Peritonite/tratamento farmacológico , Peritonite/metabolismo , Receptores de Quimiocinas/metabolismoRESUMO
When the binding of one ligand to its receptor is influenced by a second ligand acting on a different receptor, one might assume that the receptors dimerize, enabling allosteric interactions between ligands. This reasoning is frequently used to explain the complex binding curves of ligands of class A G-protein-coupled receptors (GPCRs). Here, we argue that in classical in vitro experiments the lack of GTP makes ligand-binding properties dependent on the available pool of G protein. Under such conditions a 1:1 GPCR-G-protein complex is stabilized, in which the G protein lacks a nucleotide and ligand binding is of high affinity. In vivo, this complex, a key intermediate of G-protein activation, never accumulates because of fast and irreversible GTP binding. In vitro, this complex creates interference in ligand binding when two monomeric GPCRs compete for the same G protein. Interestingly, this competition explains some in vivo effects of orphan GPCRs.
Assuntos
Receptores Acoplados a Proteínas G/fisiologia , Ligação Competitiva/fisiologia , Catálise , Dimerização , Proteínas de Ligação ao GTP/metabolismo , Humanos , CinéticaRESUMO
PURPOSE: Few reliable prognostic molecular markers have been characterized for glioblastoma multiforme (GBM), considered the deadliest of human cancers. We hypothesized that genetic polymorphisms in chemokines and their receptors, which together control microglial cell mobilization, may influence survival. METHODS: Distributions of one polymorphism of the chemokine CCL2 (-2518AAssuntos
Glioblastoma/genética
, Glioblastoma/mortalidade
, Microglia/citologia
, Polimorfismo Genético
, Receptores de Quimiocinas/genética
, Adolescente
, Adulto
, Idoso
, Feminino
, Genótipo
, Humanos
, Imuno-Histoquímica
, Masculino
, Pessoa de Meia-Idade
, Estudos Prospectivos
, Estudos Retrospectivos
RESUMO
In its native form, the chemokine CX3CL1 is a firmly adhesive molecule promoting leukocyte adhesion and migration and hence involved, along with its unique receptor CX3CR1, in various inflammatory processes. Here we investigated the role of molecular aggregation in the CX3CL1 adhesiveness. Assays of bioluminescence resonance energy transfer (BRET) and homogeneous time-resolved fluorescence (HTRF) in transfected cell lines and in primary cells showed specific signals indicative of CX3CL1 clustering. Truncation experiments showed that the transmembrane domain played a central role in this aggregation. A chimera with mutations of the 12 central transmembrane domain residues had significantly reduced BRET signals and characteristics of a non-clustering molecule. This mutant was weakly adhesive according to flow and dual pipette adhesion assays and was less glycosylated than CX3CL1, although, as we demonstrated, loss of glycosylation did not affect the CX3CL1 adhesive potency. We postulate that cell surfaces express CX3CL1 as a constitutive oligomer and that this oligomerization is essential for its adhesive potency. Inhibition of CX3CL1 self-assembly could limit the recruitment of CX3CR1-positive cells and may be a new pathway for anti-inflammatory therapies.
Assuntos
Quimiocina CX3CL1/metabolismo , Mutação , Sequência de Aminoácidos , Anti-Inflamatórios/farmacologia , Adesão Celular , Linhagem Celular , Deleção de Genes , Glicosilação , Humanos , Inflamação , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
The role of retinal microglial cells (MCs) in age-related macular degeneration (AMD) is unclear. Here we demonstrated that all retinal MCs express CX3C chemokine receptor 1 (CX3CR1) and that homozygosity for the CX3CR1 M280 allele, which is associated with impaired cell migration, increases the risk of AMD. In humans with AMD, MCs accumulated in the subretinal space at sites of retinal degeneration and choroidal neovascularization (CNV). In CX3CR1-deficient mice, MCs accumulated subretinally with age and albino background and after laser impact preceding retinal degeneration. Raising the albino mice in the dark prevented both events. The appearance of lipid-bloated subretinal MCs was drusen-like on funduscopy of senescent mice, and CX3CR1-dependent MC accumulation was associated with an exacerbation of experimental CNV. These results show that CX3CR1-dependent accumulation of subretinal MCs evokes cardinal features of AMD. These findings reveal what we believe to be a novel pathogenic process with important implications for the development of new therapies for AMD.
Assuntos
Degeneração Macular/etiologia , Microglia/patologia , Receptores de Quimiocinas/genética , Retina/patologia , Alelos , Animais , Receptor 1 de Quimiocina CX3C , Movimento Celular/genética , Neovascularização de Coroide/genética , Neovascularização de Coroide/patologia , Homozigoto , Humanos , Degeneração Macular/genética , Degeneração Macular/patologia , Masculino , Camundongos , Camundongos Knockout , Microglia/metabolismo , Polimorfismo Genético , Retina/metabolismo , Drusas Retinianas/genética , Drusas Retinianas/patologiaRESUMO
The immune system relies on the motility on various cell types that roam the host through the blood, the peripheral tissues and the lymphoid organs, looking for pathogens. Along their maturation and/or activation, the cell migratory capacities change in order to allow them to leave organs where they have been produced such as thymus and bone marrow, to locate in strategic sites to sense surrounding microbes, to meet and interact with other cells, and finally to access peripheral tissues and organs to eradicate the pathogens. This cell traffic is a highly organized process that involves numerous protein families such as adhesion molecules, proteases and chemotactic factors. Among the latter, chemokines are in the front line. We will here summarize the recent findings stressing out their physiopathological relevance and will describe thereafter their possible therapeutic use.
Assuntos
Quimiocinas/fisiologia , Migração e Rolagem de Leucócitos/fisiologia , Adjuvantes Imunológicos , Adesão Celular , Movimento Celular/fisiologia , Quimiocinas/classificação , Quimiocinas/genética , Quimiocinas/uso terapêutico , Dimerização , Endotélio Vascular/fisiologia , Glicosaminoglicanos/fisiologia , Humanos , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Polimorfismo Genético , Receptores de Quimiocinas/biossíntese , Receptores de Quimiocinas/classificação , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/fisiologia , Selectinas/fisiologia , Transdução de Sinais/fisiologiaRESUMO
The N-terminal domain of the chemokine CCL5/regulated upon activation normal T cell expressed and secreted (RANTES) has been shown to be critical for its biological activity on leukocytes. Several N-terminus-modified CCL5/RANTES derivatives, such as N-Terminal truncated CCL5/RANTES, Met-RANTES, and amino-oxypentane (AOP)-RANTES exhibited antagonist or partial agonist functions when investigated on the properties of their receptors CCR1, CCR3, and CCR5. Studying 95 African samples from Cameroon, we found a naturally occurring variant of CCL5/RANTES containing a missense mutation located in the first amino acid of the secreted form (S24F). S24F binds CCR1, CCR3, and CCR5 and triggers receptor down-modulation comparable to CCL5/RANTES. Moreover, in CCR5 positive cells, S24F elicits cellular calcium mobilization equivalent to that obtained with CCL5/RANTES. By contrast, S24F does not provoke any response in CCR1 and CCR3 positive cells. As CCL5/RANTES is able to attract different subtypes of leukocytes into inflamed tissue and intervenes in a wide range of allergic and autoimmune diseases, the discovery of this natural N-terminus-modified CCL5/RANTES analogue exhibiting differential effects on CCL5/RANTES receptors, opens up additional perspectives for therapeutic intervention.