RESUMEN
Ticks are hematophagous arachnids that parasitize mammals and other hosts, feeding on their blood. Ticks secrete numerous salivary factors that enhance host blood flow or suppress the host inflammatory response. The recruitment of leukocytes, a hallmark of inflammation, is regulated by chemokines, which activate chemokine receptors on the leukocytes. Ticks target this process by secreting glycoproteins called Evasins, which bind to chemokines and prevent leukocyte recruitment. This review describes the recent discovery of numerous Evasins produced by ticks, their classification into two structural and functional classes, and the efficacy of Evasins in animal models of inflammatory diseases. The review also proposes a standard nomenclature system for Evasins and discusses the potential of repurposing or engineering Evasins as therapeutic anti-inflammatory agents.
Asunto(s)
Quimiocinas/antagonistas & inhibidores , Proteínas de Insectos/metabolismo , Proteínas y Péptidos Salivales/metabolismo , Garrapatas/metabolismo , Animales , Leucocitos/metabolismo , Receptores de Quimiocina/metabolismo , Terminología como AsuntoRESUMEN
Recruitment of immune cells from the vasculature relies on the presentation of glycosaminoglycan-bound chemokines on the luminal side of vascular endothelial cells. However, the current model of chemokine-glycosaminoglycan interactions, and its implications for receptor interactions, remains poorly developed. We propose a refined 'Chemokine Cloud' model, arguing that chemokines are not presented to leukocytes bound to glycosaminoglycans, but rather, in solution while sequestered within the hydrated glycocalyx. We posit that glycosaminoglycans provide an immobilized chemokine depot maintaining a 'cloud' of 'solution-phase' chemokines within the glycocalyx, and that it is this soluble form of any given chemokine that interacts with leukocyte-bound receptors. Our proposition clarifies certain anomalies associated with the current model of chemokine-glycosaminoglycan interactions, with implications for the design of blockers of chemokine function.
Asunto(s)
Adhesión Celular , Leucocitos/inmunología , Leucocitos/metabolismo , Animales , Adhesión Celular/fisiología , Quimiocinas/química , Quimiocinas/metabolismo , Células Endoteliales/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Leucocitos/patología , Modelos Biológicos , Unión Proteica , Multimerización de Proteína , Receptores de Quimiocina/metabolismo , Transducción de SeñalRESUMEN
Tick evasins (EVAs) bind either CC- or CXC-chemokines by a poorly understood promiscuous or "one-to-many" mechanism to neutralize inflammation. Because EVAs potently inhibit inflammation in many preclinical models, highlighting their potential as biological therapeutics for inflammatory diseases, we sought to further unravel the CXC-chemokine-EVA interactions. Using yeast surface display, we identified and characterized 27 novel CXC-chemokine-binding evasins homologous to EVA3 and defined two functional classes. The first, which included EVA3, exclusively bound ELR+ CXC-chemokines, whereas the second class bound both ELR+ and ELR- CXC-chemokines, in several cases including CXC-motif chemokine ligand 10 (CXCL10) but, surprisingly, not CXCL8. The X-ray crystal structure of EVA3 at a resolution of 1.79 Å revealed a single antiparallel ß-sheet with six conserved cysteine residues forming a disulfide-bonded knottin scaffold that creates a contiguous solvent-accessible surface. Swapping analyses identified distinct knottin scaffold segments necessary for different CXC-chemokine-binding activities, implying that differential ligand positioning, at least in part, plays a role in promiscuous binding. Swapping segments also transferred chemokine-binding activity, resulting in a hybrid EVA with dual CXCL10- and CXCL8-binding activities. The solvent-accessible surfaces of the knottin scaffold segments have distinctive shape and charge, which we suggest drives chemokine-binding specificity. These studies provide structural and mechanistic insight into how CXC-chemokine-binding tick EVAs achieve class specificity but also engage in promiscuous binding.
Asunto(s)
Quimiocinas CXC/metabolismo , Miniproteínas Nodales de Cistina/metabolismo , Receptores de Quimiocina/metabolismo , Garrapatas/metabolismo , Animales , Cristalografía por Rayos X , Unión Proteica , Conformación Proteica , Receptores de Quimiocina/genética , Receptores de Quimiocina/aislamiento & purificación , Especificidad de la Especie , Garrapatas/clasificación , Levaduras/genéticaRESUMEN
Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZV systemic dissemination in humans.
Asunto(s)
Varicela/virología , Herpes Zóster/virología , Herpesvirus Humano 3/genética , Leucocitos/fisiología , Proteínas del Envoltorio Viral/genética , Animales , Línea Celular , Movimiento Celular , Quimiocinas/metabolismo , Varicela/inmunología , Drosophila melanogaster , Células Epiteliales/virología , Genes Reporteros , Herpes Zóster/inmunología , Herpesvirus Humano 3/inmunología , Herpesvirus Humano 3/fisiología , Interacciones Huésped-Patógeno , Humanos , Mutación , Tonsila Palatina/virología , Dominios Proteicos , Linfocitos T/virología , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , ViriónRESUMEN
Sixteen years ago, the Nomenclature Committee of the International Union of Pharmacology approved a system for naming human seven-transmembrane (7TM) G protein-coupled chemokine receptors, the large family of leukocyte chemoattractant receptors that regulates immune system development and function, in large part by mediating leukocyte trafficking. This was announced in Pharmacological Reviews in a major overview of the first decade of research in this field [Murphy PM, Baggiolini M, Charo IF, Hébert CA, Horuk R, Matsushima K, Miller LH, Oppenheim JJ, and Power CA (2000) Pharmacol Rev 52:145-176]. Since then, several new receptors have been discovered, and major advances have been made for the others in many areas, including structural biology, signal transduction mechanisms, biology, and pharmacology. New and diverse roles have been identified in infection, immunity, inflammation, development, cancer, and other areas. The first two drugs acting at chemokine receptors have been approved by the U.S. Food and Drug Administration (FDA), maraviroc targeting CCR5 in human immunodeficiency virus (HIV)/AIDS, and plerixafor targeting CXCR4 for stem cell mobilization for transplantation in cancer, and other candidates are now undergoing pivotal clinical trials for diverse disease indications. In addition, a subfamily of atypical chemokine receptors has emerged that may signal through arrestins instead of G proteins to act as chemokine scavengers, and many microbial and invertebrate G protein-coupled chemokine receptors and soluble chemokine-binding proteins have been described. Here, we review this extended family of chemokine receptors and chemokine-binding proteins at the basic, translational, and clinical levels, including an update on drug development. We also introduce a new nomenclature for atypical chemokine receptors with the stem ACKR (atypical chemokine receptor) approved by the Nomenclature Committee of the International Union of Pharmacology and the Human Genome Nomenclature Committee.
Asunto(s)
Receptores de Quimiocina , Animales , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/metabolismo , Humanos , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Receptores de Quimiocina/clasificación , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Terminología como Asunto , Garrapatas , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Interactions between chemokines such as CCL5 and glycosaminoglycans (GAGs) are essential for creating haptotactic gradients to guide the migration of leukocytes into inflammatory sites, and the GAGs that interact with CCL5 with the highest affinity are heparan sulfates/heparin. The interaction between CCL5 and its receptor on monocytes, CCR1, is mediated through residues Arg-17 and -47 in CCL5, which overlap with the GAG-binding (44)RKNR(47) "BBXB" motifs. Here we report that heparin and tetrasaccharide fragments of heparin are able to inhibit CCL5-CCR1 binding, with IC50 values showing strong dependence on the pattern and extent of sulfation. Modeling of the CCL5-tetrasaccharide complexes suggested that interactions between specific sulfate and carboxylate groups of heparin and residues Arg-17 and -47 of the protein are essential for strong inhibition; tetrasaccharides lacking the specific sulfation pattern were found to preferentially bind CCL5 in positions less favorable for inhibition of the interaction with CCR1. Simulations of a 12-mer heparin fragment bound to CCL5 indicated that the oligosaccharide preferred to interact simultaneously with both (44)RKNR(47) motifs in the CCL5 homodimer and engaged residues Arg-47 and -17 from both chains. Direct engagement of these residues by the longer heparin oligosaccharide provides a rationalization for its effectiveness as an inhibitor of CCL5-CCR1 interaction. In this mode, histidine (His-23) may contribute to CCL5-GAG interactions when the pH drops just below neutral, as occurs during inflammation. Additionally, an examination of the contribution of pH to modulating CCL5-heparin interactions suggested a need for careful interpretation of experimental results when experiments are performed under non-physiological conditions.
Asunto(s)
Quimiocina CCL5/química , Heparina/química , Oligosacáridos/química , Secuencias de Aminoácidos , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Heparina/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Oligosacáridos/metabolismo , Unión Proteica , Receptores CCR1/química , Receptores CCR1/genética , Receptores CCR1/metabolismoRESUMEN
TNF-stimulated gene/protein-6 (TSG-6) is expressed by many different cell types in response to proinflammatory cytokines and plays an important role in the protection of tissues from the damaging consequences of acute inflammation. Recently, TSG-6 was identified as being largely responsible for the beneficial effects of multipotent mesenchymal stem cells, for example in the treatment of animal models of myocardial infarction and corneal injury/allogenic transplant. The protective effect of TSG-6 is due in part to its inhibition of neutrophil migration, but the mechanisms underlying this activity remain unknown. In this study, we have shown that TSG-6 inhibits chemokine-stimulated transendothelial migration of neutrophils via a direct interaction (KD, â¼ 25 nM) between TSG-6 and the glycosaminoglycan binding site of CXCL8, which antagonizes the association of CXCL8 with heparin. Furthermore, we found that TSG-6 impairs the binding of CXCL8 to cell surface glycosaminoglycans and the transport of CXCL8 across an endothelial cell monolayer. In vivo this could limit the formation of haptotactic gradients on endothelial heparan sulfate proteoglycans and, hence, integrin-mediated tight adhesion and migration. We further observed that TSG-6 suppresses CXCL8-mediated chemotaxis of neutrophils; this lower potency effect might be important at sites where there is high local expression of TSG-6. Thus, we have identified TSG-6 as a CXCL8-binding protein, making it, to our knowledge, the first soluble mammalian chemokine-binding protein to be described to date. We have also revealed a potential mechanism whereby TSG-6 mediates its anti-inflammatory and protective effects. This could inform the development of new treatments for inflammation in the context of disease or following transplantation.
Asunto(s)
Moléculas de Adhesión Celular/inmunología , Movimiento Celular/fisiología , Interleucina-8/inmunología , Neutrófilos/inmunología , Aloinjertos , Sitios de Unión , Transporte Biológico Activo/fisiología , Adhesión Celular/fisiología , Células HL-60 , Heparina , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inflamación , Neutrófilos/citología , Trasplante de Células MadreRESUMEN
Chemokines comprise a family of secreted proteins that activate G protein-coupled chemokine receptors and thereby control the migration of leukocytes during inflammation or immune surveillance. The positional information required for such migratory behavior is governed by the binding of chemokines to membrane-tethered glycosaminoglycans (GAGs), which establishes a chemokine concentration gradient. An often observed but incompletely understood behavior of chemokines is the ability of unrelated chemokines to enhance the potency with which another chemokine subtype can activate its cognate receptor. This phenomenon has been demonstrated to occur between many chemokine combinations and across several model systems and has been dubbed chemokine cooperativity. In this study, we have used GAG binding-deficient chemokine mutants and cell-based functional (migration) assays to demonstrate that chemokine cooperativity is caused by competitive binding of chemokines to GAGs. This mechanistic explanation of chemokine cooperativity provides insight into chemokine gradient formation in the context of inflammation, in which multiple chemokines are secreted simultaneously.
Asunto(s)
Quimiocinas/metabolismo , Glicosaminoglicanos/metabolismo , Animales , Unión Competitiva , Células CHO , Quimiocina CCL19/metabolismo , Quimiocina CCL21/metabolismo , Quimiocina CXCL13/metabolismo , Quimiocinas/química , Quimiotaxis , Cricetinae , Cricetulus , Modelos Biológicos , Unión Proteica , Multimerización de Proteína , Receptores de Quimiocina/metabolismoRESUMEN
To elucidate the ligand-binding surface of the CC chemokine-binding proteins Evasin-1 and Evasin-4, produced by the tick Rhipicephalus sanguineus, we sought to identify the key determinants responsible for their different chemokine selectivities by expressing Evasin mutants using phage display. We first designed alanine mutants based on the Evasin-1·CCL3 complex structure and an in silico model of Evasin-4 bound to CCL3. The mutants were displayed on M13 phage particles, and binding to chemokine was assessed by ELISA. Selected variants were then produced as purified proteins and characterized by surface plasmon resonance analysis and inhibition of chemotaxis. The method was validated by confirming the importance of Phe-14 and Trp-89 to the inhibitory properties of Evasin-1 and led to the identification of a third crucial residue, Asn-88. Two amino acids, Glu-16 and Tyr-19, were identified as key residues for binding and inhibition of Evasin-4. In a parallel approach, we identified one clone (Y28Q/N60D) that showed a clear reduction in binding to CCL3, CCL5, and CCL8. It therefore appears that Evasin-1 and -4 use different pharmacophores to bind CC chemokines, with the principal binding occurring through the C terminus of Evasin-1, but through the N-terminal region of Evasin-4. However, both proteins appear to target chemokine N termini, presumably because these domains are key to receptor signaling. The results also suggest that phage display may offer a useful approach for rapid investigation of the pharmacophores of small inhibitory binding proteins.
Asunto(s)
Quimiocinas CC/química , Receptores de Quimiocina/química , Alanina/química , Secuencia de Aminoácidos , Animales , Movimiento Celular , Quimiocina CCL3/química , Quimiocina CCL5/química , Quimiocina CCL5/genética , Quimiocina CCL8/química , Quimiotaxis , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Glicosilación , Células HEK293 , Humanos , Ligandos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Biblioteca de Péptidos , Unión Proteica , Estructura Terciaria de Proteína , Rhipicephalus sanguineus , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de SuperficieRESUMEN
Chemoattractant cytokines, or chemokines, are the largest sub-family of cytokines. About 50 distinct chemokines have been identified in humans. Their principal role is to stimulate the directional migration of leukocytes, which they achieve through activation of their receptors, following immobilization on cell surface glycosaminoglycans (GAGs). Chemokine receptors belong to the G protein-coupled 7-transmembrane receptor family, and hence their identification brought great promise to the pharmaceutical industry, since this receptor class is the target for a large percentage of marketed drugs. Unfortunately, the development of potent and efficacious inhibitors of chemokine receptors has not lived up to the early expectations. Several approaches to targeting this system will be described here, which have been instrumental in establishing paradigms in chemokine biology. Whilst drug discovery programs have not yet elucidated how to make successful drugs targeting the chemokine system, it is now known that certain parasites have evolved anti-chemokine strategies in order to remain undetected by their hosts. What can we learn from them?
Asunto(s)
Quimiocinas/antagonistas & inhibidores , Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Receptores de Quimiocina/antagonistas & inhibidores , Animales , Quimiocinas/inmunología , Humanos , Receptores de Quimiocina/inmunologíaRESUMEN
Chemokines and their receptors are involved in the pathogenesis of diseases ranging from asthma to AIDS. Chemokine receptors are G-protein-coupled serpentine receptors that present attractive tractable targets for the pharmaceutical industry. It is only ten years since the first chemokine receptor was discovered, and the rapidly expanding number of antagonists holds promise for new medicines to combat diseases that are currently incurable. Here, I focus on the rationale for developing antagonists of chemokine receptors for inflammatory disorders and AIDS, and the accumulating evidence that favours this strategy despite the apparent redundancy in the chemokine system.
Asunto(s)
Receptores de Quimiocina/antagonistas & inhibidores , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Animales , Quimiocinas/antagonistas & inhibidores , Quimiocinas/fisiología , Modelos Animales de Enfermedad , Humanos , Inflamación/tratamiento farmacológico , Ratones , Ratones Noqueados , Receptores de Quimiocina/fisiologíaRESUMEN
Neutrophils are rapidly and massively recruited to sites of microbial infection, where they can influence the recruitment of dendritic cells. Here, we have analyzed the role of neutrophil released chemokines in the early recruitment of dendritic cells (DCs) in an experimental model of Leishmania major infection. We show in vitro, as well as during infection, that the parasite induced the expression of CCL3 selectively in neutrophils from L. major resistant mice. Neutrophil-secreted CCL3 was critical in chemotaxis of immature DCs, an effect lost upon CCL3 neutralisation. Depletion of neutrophils prior to infection, as well as pharmacological or genetic inhibition of CCL3, resulted in a significant decrease in DC recruitment at the site of parasite inoculation. Decreased DC recruitment in CCL3(-/-) mice was corrected by the transfer of wild type neutrophils at the time of infection. The early release of CCL3 by neutrophils was further shown to have a transient impact on the development of a protective immune response. Altogether, we identified a novel role for neutrophil-secreted CCL3 in the first wave of DC recruitment to the site of infection with L. major, suggesting that the selective release of neutrophil-secreted chemokines may regulate the development of immune response to pathogens.
Asunto(s)
Quimiocina CCL3/inmunología , Quimiotaxis de Leucocito/inmunología , Células Dendríticas/inmunología , Leishmaniasis Cutánea/inmunología , Neutrófilos/inmunología , Animales , Quimiocina CCL3/metabolismo , Células Dendríticas/metabolismo , Femenino , Citometría de Flujo , Leishmania major/inmunología , Leishmaniasis Cutánea/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neutrófilos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Chemokine-glycosaminoglycan (GAG) interactions are thought to result in the formation of tissue-bound chemokine gradients. We hypothesized that the binding of chemokines to GAGs would increase neutrophil migration toward CXC chemokines instilled into lungs of mice. To test this hypothesis we compared neutrophil migration toward recombinant human CXCL8 (rhCXCL8) and two mutant forms of CXCL8, which do not bind to heparin immobilized on a sensor chip. Unexpectedly, when instilled into the lungs of mice the CXCL8 mutants recruited more neutrophils than rhCXCL8. The CXCL8 mutants appeared in plasma at significantly higher concentrations and diffused more rapidly across an extracellular matrix in vitro. A comparison of the murine CXC chemokines, KC and MIP-2, revealed that KC was more effective in recruiting neutrophils into the lungs than MIP-2. KC appeared in plasma at significantly higher concentrations and diffused more rapidly across an extracellular matrix in vitro than MIP-2. In kinetic binding studies, KC, MIP-2, and rhCXCL8 bound heparin differently, with KC associating and dissociating more rapidly from immobilized heparin than the other chemokines. These data suggest that the kinetics of chemokine-GAG interactions contributes to chemokine function in tissues. In the lungs, it appears that chemokines, such as CXCL8 or MIP-2, which associate and disassociate slowly from GAGs, form gradients relatively slowly compared with chemokines that either bind GAGs poorly or interact with rapid kinetics. Thus, different types of chemokine gradients may form during an inflammatory response. This suggests a new model, whereby GAGs control the spatiotemporal formation of chemokine gradients and neutrophil migration in tissue.
Asunto(s)
Movimiento Celular , Quimiocinas/metabolismo , Glicosaminoglicanos/metabolismo , Pulmón/metabolismo , Neutrófilos/metabolismo , Animales , Células CHO , Quimiocina CXCL2/metabolismo , Quimiotaxis de Leucocito , Cricetinae , Cricetulus , Citometría de Flujo , Heparina/metabolismo , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Cinética , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Neutrófilos/citología , Unión Proteica , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
CCL3 is a protein of the CC chemokine family known to be important for T cell recruitment in inflammatory diseases. The aim of the current study was to evaluate the effects and putative mechanism of action of evasin-1, a novel CCL3-binding protein, in the pathogenesis of acute graft-versus-host disease (GVHD). GVHD was induced by the transplantation of splenocytes from C57BL/6J to B6D2F1 mice. Treatment of recipient mice with evasin-1 prevented mortality associated with GVHD. This was correlated with reduced weight loss and clinical disease severity. Analysis of the small intestine showed that evasin-1 treatment reduced the histopathological score and decreased levels of IFN-gamma and CCL5. Mechanistically, evasin-1 treatment reduced the number of CD4(+) and CD8(+) T cells infiltrating the small intestine, as assessed by immunohistochemistry, and the adhesion of leukocytes to intestinal venules of recipient mice, as assessed by intravital microscopy. Evasin-1 was also able to decrease liver damage, as seen by reduction of inflammatory infiltrate and IFN-gamma levels. Treatment with evasin-1 did not interfere with graft-versus-leukemia. Altogether, our studies demonstrate that CCL3 plays a major role in mediating GVHD, but not graft-versus-leukemia in mice and suggest that blockade of CCL3 with evasin-1 has potential therapeutic application in patients undergoing bone marrow transplantation.
Asunto(s)
Quimiocina CCL3/deficiencia , Enfermedad Injerto contra Huésped/metabolismo , Proteínas Inflamatorias de Macrófagos/metabolismo , Receptores de Quimiocina/metabolismo , Animales , Antiinflamatorios/metabolismo , Antiinflamatorios/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Trasplante de Células , Quimiocina CCL3/genética , Quimiocina CCL5/metabolismo , Dexametasona/farmacología , Femenino , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/prevención & control , Efecto Injerto vs Leucemia/efectos de los fármacos , Inmunohistoquímica , Interferón gamma/metabolismo , Intestino Delgado/inmunología , Intestino Delgado/metabolismo , Intestino Delgado/patología , Proteínas Inflamatorias de Macrófagos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos , Ratones Transgénicos , Bazo/citologíaRESUMEN
CC chemokines play an important role in the pathogenesis of idiopathic pulmonary fibrosis. Few studies have evaluated the efficacy of therapeutically targeting CC chemokines and their receptors during interstitial lung diseases. In the present study, the therapeutic effects of Evasin-1, a tick-derived chemokine-binding protein that has high affinity for CCL3/microphage inflammatory protein (MIP)-1α, was investigated in a murine model of bleomycin-induced lung fibrosis. CCL3/MIP-1α concentrations in lung homogenates increased significantly with time after bleomycin challenge, and this was accompanied by increased number of leukocytes and elevated levels of CCL2/monocyte chemoattractant protein (MCP)-1, CCL5/regulated upon activation, normal T cell expressed and secreted, TNF-α and transforming growth factor-ß(1), and pulmonary fibrosis. Administration of evasin-1 on a preventive (from the day of bleomycin administration) or therapeutic (from Day 8 after bleomycin) schedule decreased number of leukocytes in the lung, reduced levels of TNF-α and transforming growth factor-ß(1), and attenuated lung fibrosis. These protective effects were similar to those observed in CCL3/MIP-1α-deficient mice. In conclusion, targeting CCL3/MIP-1α by treatment with evasin-1 is beneficial in the context of bleomycin-induced lung injury, even when treatment is started after the fibrogenic insult. Mechanistically, evasin-1 treatment was associated with decreased recruitment of leukocytes and production of fibrogenic cytokines. Modulation of CCL3/MIP-1α function by evasin-1 could be useful for the treatment of idiopathic pulmonary fibrosis.
Asunto(s)
Antibióticos Antineoplásicos/efectos adversos , Bleomicina/efectos adversos , Quimiocina CCL3/antagonistas & inhibidores , Quimiocina CCL3/inmunología , Fibrosis Pulmonar/tratamiento farmacológico , Receptores de Quimiocina/uso terapéutico , Animales , Antibióticos Antineoplásicos/administración & dosificación , Bleomicina/farmacología , Quimiocina CCL3/genética , Quimiocina CCL3/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Leucocitos/inmunología , Leucocitos/metabolismo , Leucocitos/patología , Masculino , Ratones , Ratones Noqueados , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/inmunología , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Receptores de Quimiocina/química , Rhipicephalus sanguineus/química , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Chemokines orchestrate the migration of leukocytes in the context of homeostasis and inflammation. In addition to interactions of chemokines with receptors on migrating cells, these processes require interactions of chemokines with glycosaminoglycans (GAGs) for cell surface localization. Most chemokines are basic proteins with Arg/Lys/His residue clusters functioning as recognition epitopes for GAGs. In this study we characterized the GAG-binding epitopes of the chemokine I-TAC/CXCL11. Four separate clusters of basic residues were mutated to alanine and tested for their ability to bind to GAGs in vitro and to activate the receptor, CXCR3. Mutation of a set of basic residues in the C-terminal helix (the 50s cluster, (57)KSKQAR(62)) along with Lys(17), significantly impaired heparin binding in vitro, identifying these residues as components of the dominant epitope. However, this GAG mutant retained nearly wild type receptor binding affinity, and its ability to induce cell migration in vitro was only mildly perturbed. Nevertheless, the mutant was unable to induce cell migration in vivo, establishing a requirement of CXCL11 for GAG binding for in vivo function. These studies also led to some interesting findings. First, CXCL11 exhibits conformational heterogeneity, as evidenced by the doubling of peaks in its HSQC spectra. Second, it exhibits more than one affinity state for both heparin and CXCR3, which may be related to its structural plasticity. Finally, although the binding affinities of chemokines for GAGs are typically weaker than interactions with receptors, the high affinity GAG binding state of CXCL11 is comparable with typical receptor binding affinities, suggesting some unique properties of this chemokine.
Asunto(s)
Quimiocina CXCL11/metabolismo , Glicosaminoglicanos/química , Heparina/química , Alanina/química , Animales , Sitios de Unión , Movimiento Celular , Epítopos/química , Femenino , Ratones , Ratones Endogámicos BALB C , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Receptores CXCR3/química , Células TH1/metabolismoRESUMEN
Eosinophils are multifunctional leukocytes implicated in numerous inflammatory diseases. The present study was conducted to clarify the precise role of eosinophils in the development of colitis by using eosinophil-depleted mice and a novel chemokine-binding protein that neutralizes CCL11 action. Colitis was induced by administration of dextran sodium sulfate (DSS) to wild-type and eosinophil-deficient DeltadblGATA-1 mice. Accumulation of eosinophils in the gut of mice given DSS paralleled worsening of clinical score and weight loss. In response to DSS, DeltadblGATA-1 mice showed virtual absence of eosinophil recruitment, amelioration of clinical score, weight loss, and tissue destruction, and no lethality. There was a decrease in CXCL1 and CCL3 production and decreased neutrophil influx in the intestine of DeltadblGATA-1 mice. Transfer of bone marrow cells from wild-type mice reconstituted disease manifestation in DSS-treated DeltadblGATA-1 mice, and levels of CCL11 were increased after DSS treatment and localized to inflammatory cells. Treatment with the chemokine-binding protein evasin-4 at a dose that prevented the function of CCL11 greatly ameliorated clinical score, weight loss, overall tissue destruction, and death rates. In conclusion, the influx of eosinophils is critical for the induction of colitis by DSS. Treatment with a novel chemokine-binding protein decreased eosinophil influx and greatly ameliorated colitis, suggesting that strategies that interfere with the recruitment of eosinophils may be useful as therapy for colitis.
Asunto(s)
Quimiocina CCL11/inmunología , Colitis/inmunología , Eosinófilos/inmunología , Animales , Linaje de la Célula , Inhibición de Migración Celular/inmunología , Factores Quimiotácticos Eosinófilos/antagonistas & inhibidores , Quimiotaxis de Leucocito/inmunología , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB CRESUMEN
OBJECTIVE: We examined the potential contribution of CCL3 and CCL5 to inflammatory angiogenesis in mice. METHODS: Polyester-polyurethane sponges were implanted in mice and blood vessel counting and hemoglobin, myeloperoxidase and N-acetylglucosaminidase measurements used as indexes for vascularization, neutrophil and macrophage accumulation, respectively. RESULTS: CCL3 and CCL5 were expressed throughout the observation period. Exogenous CCL3 enhanced angiogenesis in WT, but angiogenesis proceeded normally in CCL3(-/-) mice, suggesting that endogenous CCL3 is not critical for sponge-induced angiogenesis in mice. CCL5 expression was detected at day 1, but levels significantly increased thereafter. Exogenous CCL5 reduced angiogenesis in WT mice possible via CCR5 as CCL5 was without an effect in CCR5(-/-) mice. Treatment of WT with the CCR1/CCR5 antagonist, Met-RANTES, prevented neutrophil and macrophage accumulation, but enhanced sponge vascularization. CONCLUSION: Thus, endogenous CCL3 appears not to play a role in driving sponge-induced inflammatory angiogenesis in mice. The effects of CCL5 were anti-angiogenic and appeared to be mediated via activation of CCR5.
Asunto(s)
Quimiocina CCL3/metabolismo , Quimiocina CCL5/metabolismo , Inflamación/inmunología , Neovascularización Fisiológica , Tapones Quirúrgicos de Gaza/efectos adversos , Animales , Quimiocina CCL3/genética , Quimiocina CCL5/antagonistas & inhibidores , Quimiocina CCL5/genética , Quimiocina CCL5/farmacología , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Recombinantes/metabolismo , Factores de TiempoRESUMEN
We demonstrated recently that P8A-CCL2, a monomeric variant of the chemokine CCL2/MCP-1, is unable to induce cellular recruitment in vivo, despite full activity in vitro. Here, we show that this variant is able to inhibit CCL2 and thioglycollate-mediated recruitment of leukocytes into the peritoneal cavity and recruitment of cells into lungs of OVA-sensitized mice. This anti-inflammatory activity translated into a reduction of clinical score in the more complex inflammatory model of murine experimental autoimmune encephalomyelitis. Several hypotheses for the mechanism of action of P8A-CCL2 were tested. Plasma exposure following s.c. injection is similar for P8A-CCL2 and wild-type (WT) CCL2, ruling out the hypothesis that P8A-CCL2 disrupts the chemokine gradient through systemic exposure. P8A-CCL2 and WT induce CCR2 internalization in vitro and in vivo; CCR2 then recycles to the cell surface, but the cells remain refractory to chemotaxis in vitro for several hours. Although the response to P8A-CCL2 is similar to WT, this finding is novel and suggests that despite the presence of the receptor on the cell surface, coupling to the signaling machinery is retarded. In contrast to CCL2, P8A-CCL2 does not oligomerize on glycosaminoglycans (GAGs). However, it retains the ability to bind GAGs and displaces endogenous JE (murine MCP-1) from endothelial surfaces. Intravital microscopy studies indicate that P8A-CCL2 prevents leukocyte adhesion, while CCL2 has no effect, and this phenomenon may be related to the mechanism. These results suggest that oligomerization-deficient chemokines can exhibit anti-inflammatory properties in vivo and may represent new therapeutic modalities.
Asunto(s)
Antiinflamatorios/farmacología , Quimiocina CCL2/farmacología , Quimiocinas/fisiología , Leucocitos/fisiología , Animales , Encéfalo/citología , Circulación Cerebrovascular/fisiología , Quimiocina CCL2/genética , Quimiocinas/química , Quimiotaxis de Leucocito , Femenino , Ingeniería Genética , Heparina/farmacología , Inmunización , Leucocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Tioglicolatos/farmacologíaRESUMEN
Multiple sclerosis is believed to be an autoimmune disease with an end-point of neuro-degeneration, but in which inflammation plays a predominant role. Therefore therapies which target inhibition of the excessive recruitment of leukocytes into the central nervous system (CNS) are actively sought after by medical research. Drug discovery relies heavily on animal models used for such research, called Experimental Autoimmune Encephalomyelitis (EAE). Several chemokines and their receptors have been shown to play a role in this recruitment into the CNS, and we have investigated several strategies which antagonize this system in EAE models. We will discuss these strategies and their successes and failures to prevent disease symptoms and the insights they have provided.