RESUMEN

Chemokines are essential for homeostasis and activation of the immune system. The chemokine ligand/receptor pairing CCL20/CCR6 is interesting because these molecules display characteristics of both homeostatic and activation functions. These dual characteristics suggest a role for CCR6 in the priming and effector phases of the immune response. However, while CCR6 has been implicated in the effector phase in several models, a role in the priming phase is less clear. Herein we analyze the role of CCR6 in these two important arms of the immune response during experimental autoimmune encephalomyelitis (EAE). Both CCR6 and its chemokine ligand CCL20 were up-regulated in the draining lymph nodes and spinal cord during EAE, and CCR6 was up-regulated on CD4(+) T cells that had divided following induction of EAE. The functional role of this expression was demonstrated by impaired development of EAE in gene-targeted CCR6-deficient mice and in mice treated either with a neutralizing anti-CCR6 Ab or with a novel receptor antagonist. Inhibition of EAE was due to reduced priming of autoreactive CD4(+) T cells probably as a result of impaired late-stage influx of dendritic cells into draining lymph nodes. This was accompanied by reduced egress of activated lymphocytes from the lymph nodes. These results demonstrate a novel role for CCR6 in the mechanism of autoreactive lymphocyte priming and emigration to the efferent lymphatics.

Asunto(s)

Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/terapia , Receptores CCR6/antagonistas & inhibidores , Receptores CCR6/fisiología , Secuencia de Aminoácidos , Animales , Quimiocina CCL20/biosíntesis , Quimiocina CCL20/genética , Quimiocina CCL20/fisiología , Quimiotaxis de Leucocito/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/patología , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Vasos Linfáticos/inmunología , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Receptores CCR6/biosíntesis , Índice de Severidad de la Enfermedad , Médula Espinal/inmunología , Médula Espinal/metabolismo , Médula Espinal/patología

RESUMEN

Chemokines regulate lymphocyte trafficking under physiologic and pathologic conditions. In this study, we have investigated the role of CXCR3 and CXCR4 in the activation of T lymphocytes and their migration to the central nervous system (CNS) using novel mutant chemokines to antagonize CXCR3 and CXCR4 specifically. A series of truncation mutants of CXCL11, which has the highest affinity for CXCR3, were synthesized, and an antagonist, CXCL11((4-79)), was obtained. CXCL11((4-79)) strongly inhibited the migration of activated mouse T cells in response to all three high-affinity CXCR3 ligands, CXCL9, 10 and 11. CXCL12((P2G2)), while exhibiting minimal agonistic activity, potently inhibited the migration of activated mouse T cells in response to CXCL12. Interfering with the action of CXCR3 and CXCR4 with these synthetic receptor antagonists inhibited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis and reduced the accumulation of CD4(+) T cells in the CNS. Further investigation demonstrated that CXCL12((P2G2)) inhibited the sensitization phase, whereas CXCL11((4-79)) inhibited the effector phase of the immune response. Our data suggest that simultaneous targeting of CXCR4 and CXCR3 may be of benefit in the treatment of the CNS autoimmune disease.

Asunto(s)

Sistema Nervioso Central/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Factores Inmunológicos/farmacología , Receptores CXCR3/antagonistas & inhibidores , Receptores CXCR4/antagonistas & inhibidores , Traslado Adoptivo , Animales , Células Cultivadas , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/fisiopatología , Quimiocina CXCL11/antagonistas & inhibidores , Quimiocina CXCL11/genética , Quimiocina CXCL11/inmunología , Quimiocina CXCL12/antagonistas & inhibidores , Quimiocina CXCL12/genética , Quimiocina CXCL12/inmunología , Quimiocinas/agonistas , Quimiocinas/genética , Quimiocinas/inmunología , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/inmunología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Factores Inmunológicos/uso terapéutico , Terapia de Inmunosupresión/métodos , Ratones , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/inmunología , Péptidos/farmacología , Receptores CXCR3/inmunología , Receptores CXCR4/inmunología , Homología de Secuencia de Aminoácido , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Resultado del Tratamiento

RESUMEN

Interleukin-8 (IL-8), a member of the chemokine superfamily, exists as both monomers and dimers, and mediates its function by binding to neutrophil CXCR1 and CXCR2 receptors that belong to the G protein-coupled receptor class. It is now well established that the monomer functions as a high-affinity ligand, but the binding affinity of the dimer remains controversial. The approximately 1000-fold difference between monomer-dimer equilibrium constant (microM) and receptor binding constant (nM) of IL-8 does not allow receptor-binding affinity measurements of the native IL-8 dimer. In this study, we overcame this roadblock by creating a "trapped" nondissociating dimer that contains a disulfide bond across the dimer interface at the 2-fold symmetry point. The NMR studies show that the structure of this trapped dimer is indistinguishable from the native dimer. The trapped dimer, compared to a trapped monomer, bound CXCR1 with approximately 70-fold and CXCR2 with approximately 20-fold lower affinities. Receptor binding involves two interactions, between the IL-8 N-loop and receptor N-domain residues, and between IL-8 N-terminal and receptor extracellular loop residues. In contrast to a trapped monomer that bound an isolated CXCR1 N-domain peptide with microM affinity, the trapped dimer failed to show any binding, indicating that dimerization predominantly perturbs the binding of only the N-loop residues. These results demonstrate that only the monomer is a high-affinity ligand for both receptors, and also provide a structural basis for the lower binding affinity of the dimer.

Asunto(s)

Disulfuros/química , Interleucina-8/química , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/metabolismo , Sustitución de Aminoácidos , Dimerización , Interleucina-8/metabolismo , Resonancia Magnética Nuclear Biomolecular

RESUMEN

During polyketide biosynthesis, malonyl groups are transferred to the acyl carrier protein (ACP) component of the polyketide synthase (PKS), and it has been shown that a number of type II polyketide ACPs undergo rapid self-acylation from malonyl-CoA in the absence of a malonyl-CoA:holo-acyl carrier protein transacylase (MCAT). More recently, however, the observation of self-malonylation has been ascribed to contamination with Escherichia coli MCAT (FabD) rather than an intrinsic property of the ACP. The wild-type apo-ACP from the actinorhodin (act) PKS of Streptomyces coelicolor (synthetic apo-ACP) has therefore been synthesized using solid-state peptide methods and refolded using the GroEL/ES chaperone system from E. coli. Correct folding of the act ACP has been confirmed by circular dichroism (CD) and 1H NMR. Synthetic apo-ACP was phosphopantetheinylated to 100% by S. coelicolor holo-acyl carrier protein synthase (ACPS), and the resultant holo-ACP underwent self-malonylation in the presence of malonyl-CoA. No malonylation of negative controls was observed, confirming that the use of ACPS and GroEL/ES did not introduce contamination with E. coli MCAT. This result proves unequivocally that self-malonylation is an inherent activity of this PKS ACP in vitro.

Asunto(s)

Proteína Transportadora de Acilo/síntesis química , Proteína Transportadora de Acilo/metabolismo , Malonatos/metabolismo , Sintasas Poliquetidas/metabolismo , S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo/metabolismo , Apoproteínas/síntesis química , Apoproteínas/metabolismo , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Resonancia Magnética Nuclear Biomolecular , Pliegue de Proteína , Espectrometría de Masa por Ionización de Electrospray , Streptomyces coelicolor/enzimología

RESUMEN

The foreign body reaction (FBR) develops in response to the implantation of almost all biomaterials and can be detrimental to their function. The formation of foreign body giant cells (FBGC), which damage the surface of biomaterials, is considered a hallmark of this reaction. FBGC derive from blood-borne monocytes that enter the implantation site after surgery in response to the release of chemotactic signals. In this study, we implanted biomaterials subcutaneous (s.c.) in mice that lack the monocyte chemoattractant CC chemokine ligand 2 (CCL2) and found that biomaterials were encapsulated despite reduced FBGC formation. The latter was due to compromised macrophage fusion rather than migration. Consistent with the reduction in FBGC formation, biodegradable biomaterials sustained reduced damage in CCL2-null mice. Furthermore, blockade of CCL2 function by localized gene delivery in wild-type mice hindered FBGC formation, despite normal monocyte recruitment. The requirement for CCL2 in fusion was confirmed by the ability of both a CCL2 inhibitory peptide and an anti-CCL2 Ab to reduce FBGC formation from peripheral blood monocytes in an in vitro assay. Our findings demonstrate a previously unreported involvement of CCL2 in FBGC formation, and suggest that FBGC are not the primary determinants of capsule formation in the FBR.

Asunto(s)

Quimiocina CCL2/fisiología , Células Gigantes de Cuerpo Extraño/metabolismo , Células Gigantes de Cuerpo Extraño/patología , Macrófagos/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Materiales Biocompatibles/administración & dosificación , Fusión Celular , Movimiento Celular , Quimiocina CCL2/genética , Quimiocina CCL2/inmunología , Quimiocinas CC/metabolismo , Femenino , Ligandos , Macrófagos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/citología , Monocitos/metabolismo , Fragmentos de Péptidos/farmacología

RESUMEN

While chemokines are clearly important in the generation of protective immunity, the role of individual chemokines in the control of bacterial infection is still poorly understood. In this study, we investigated the role of macrophage inflammatory protein (MIP)-3alpha/CCL20, a chemokine that attracts activated T and B lymphocytes and immature dendritic cells, in host responses to bacterial infection. CCL20 production was induced in subcutaneous tissue in the BALB/c mouse in response to Salmonella enteritidis, Staphylococcus aureus and zymosan, with S. enteritidis being the most potent. S. enteritidis induced CCL20 production in the spleen following either oral administration or injection into the peritoneal cavity. In contrast, no increase was observed in the Peyer's patches. In this model, following intraperitoneal injection, dose-dependent colonization of the spleen and Peyer's patches by S. enteritidis, expression of IFNgamma and IL-4, and production of antibodies against the S. enteritidis surface antigen SefA were observed. Prior treatment with neutralizing antibodies against CCL20 enhanced bacterial dissemination to the spleen and Peyer's patches and strongly biased the IFNgamma/IL-4 ratio towards a type 2 profile in the spleen, while the humoral response was unaffected. In contrast, treatment with neutralizing anti-MIP-1alpha/CCL3 antibodies enhanced the bacterial burden in the Peyer's patches but not in the spleen, had no significant effect on the cytokine ratio, but significantly inhibited anti-SefA production. Together, these results demonstrate an important role for CCL20 in the control of bacterial infection and more specifically in the regulation of cell-mediated immunity against intracellular bacteria such as S. enteritidis.

Asunto(s)

Quimiocinas CC/fisiología , Proteínas Inflamatorias de Macrófagos/fisiología , Infecciones por Salmonella/microbiología , Salmonella enteritidis/crecimiento & desarrollo , Animales , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Quimiocina CCL20 , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocinas CC/inmunología , Recuento de Colonia Microbiana , Relación Dosis-Respuesta Inmunológica , Femenino , Proteínas Fimbrias/inmunología , Proteínas Fimbrias/metabolismo , Interferón gamma/biosíntesis , Interleucina-4/biosíntesis , Proteínas Inflamatorias de Macrófagos/inmunología , Proteínas Inflamatorias de Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Cavidad Peritoneal/microbiología , Ganglios Linfáticos Agregados/inmunología , Ganglios Linfáticos Agregados/microbiología , Infecciones por Salmonella/inmunología , Salmonella enteritidis/inmunología , Bazo/inmunología , Bazo/metabolismo , Bazo/microbiología , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/inmunología

RESUMEN

Chemokines constitute a group of over 40 secreted peptides that are important for the control of leukocyte migration both during homeostasis and inflammation. Recent studies have implicated the ligands CCL19 and CCL21 and their receptor, CCR7, in the specific migration of naïve lymphocytes and mature dendritic cells to secondary lymphoid organs during immune homeostasis. However, the role that these molecules play during immune priming is not well understood. In this study, using CCL19((8-83)), a novel N-terminal truncation mutant, we have investigated the role of CCL19 in a primary allogeneic immune response, a response of particular relevance to transplant rejection. This antagonist specifically inhibited wild type CCL19-induced chemotaxis and intracellular calcium mobilization without affecting that of CCL21. The treatment of mice with CCL19((8-83)) did not globally inhibit the recruitment of cells into lymph nodes; however, it inhibited the generation of cytotoxic T lymphocytes toward allogeneic dendritic cells. This is the first evidence that CCL19 plays a role in immune priming.

Asunto(s)

Quimiocinas CC/antagonistas & inhibidores , Quimiocinas CC/metabolismo , Linfocitos T Citotóxicos/metabolismo , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Movimiento Celular , Quimiocina CCL19 , Quimiocina CCL21 , Quimiocinas/metabolismo , Quimiotaxis , Células Dendríticas/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Sistema Inmunológico , Inflamación , Leucocitos/metabolismo , Ligandos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutación , Péptidos/química , Unión Proteica , Estructura Terciaria de Proteína , Receptores CCR7 , Receptores de Quimiocina/metabolismo , Bazo/citología , Factores de Tiempo

RESUMEN

CXCL11 (ITAC) is one of three chemokines known to bind the receptor CXCR3, the two others being CXCL9 (Mig) and CXCL10 (IP-10). CXCL11 differs from the other CXCR3 ligands in both the strength and the particularities of its receptor interactions: It has a higher affinity, is a stronger agonist, and behaves differently when critical N-terminal residues are deleted. The structure of CXCL11 was determined using solution NMR to allow comparison with that of CXCL10 and help elucidate the source of the differences. CXCL11 takes on the canonical chemokine fold but exhibits greater conformational flexibility than has been observed for related chemokines under the same sample conditions. Unlike related chemokines such as IP-10 and IL-8, ITAC does not appear to form dimers at millimolar concentrations. The origin for this behavior can be found in the solution structure, which indicates a beta-bulge in beta-strand 1 that distorts the dimerization interface used by other CXC chemokines.

Asunto(s)

Quimiocinas CXC/química , Receptores de Quimiocina/química , Quimiocina CXCL11 , Quimiocinas CXC/metabolismo , Dimerización , Humanos , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores CXCR3 , Receptores de Quimiocina/metabolismo

RESUMEN

The mechanisms of neurodegeneration that result in human immunodeficiency virus (HIV) type 1 dementia have not yet been identified. Here, we report that HIV-infected macrophages secrete the zymogen matrix metalloproteinase-2 (MMP-2), which is activated by exposure to MT1-MMP on neurons. Stromal cell-derived factor 1 alpha (SDF-1), a chemokine overexpressed by astrocytes during HIV infection, was converted to a highly neurotoxic protein after precise proteolytic processing by active MMP-2, which removed the N-terminal tetrapeptide. Implantation of cleaved SDF-1(5-67) into the basal ganglia of mice resulted in neuronal death and inflammation with ensuing neurobehavioral deficits that were abrogated by neutralizing antibodies to SDF-1 and an MMP inhibitor drug. Hence, this study identifies a new in vivo neurotoxic pathway in which cleavage of a chemokine by an induced metalloproteinase results in neuronal apoptosis that leads to neurodegeneration.

Asunto(s)

Complejo SIDA Demencia/enzimología , Quimiocinas CXC/toxicidad , Metaloproteinasa 2 de la Matriz/metabolismo , Degeneración Nerviosa/enzimología , Neurotoxinas/toxicidad , Complejo SIDA Demencia/etiología , Complejo SIDA Demencia/fisiopatología , Animales , Anticuerpos/farmacología , Astrocitos/metabolismo , Línea Celular , Quimiocina CXCL12 , Quimiocinas CXC/antagonistas & inhibidores , Quimiocinas CXC/metabolismo , Modelos Animales de Enfermedad , Encefalitis/inducido químicamente , Encefalitis/enzimología , Encefalitis/fisiopatología , Inhibidores Enzimáticos/farmacología , VIH-1/metabolismo , VIH-1/patogenicidad , Humanos , Macrófagos/enzimología , Macrófagos/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Neostriado/efectos de los fármacos , Neostriado/patología , Neostriado/fisiopatología , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/virología , Neurotoxinas/metabolismo , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad

RESUMEN

Viruses encode proteins that disrupt chemokine responses. The murine gammaherpesvirus 68 gene M3 encodes a chemokine binding protein (vCKBP-3) which has no sequence similarity to chemokine receptors but inhibits chemokine receptor binding and activity. We have used a panel of CXCL8 analogs to identify the structural requirements for CXCL8 to bind to vCKBP-3 in a scintillation proximity assay. Our data suggest that vCKBP-3 acts by mimicking the binding of chemokine receptors to CXCL8.

Asunto(s)

Quimiocinas CXC/química , Quimiocinas CXC/metabolismo , Gammaherpesvirinae/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Quimiocinas CXC/genética , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica

RESUMEN

Chemokines are a family of cytokines that exhibit selective chemoattractant properties for target leukocytes and play a significant role in leukocyte migration. In this study, we have investigated the role of the C-C chemokine, macrophage inflammatory protein (MIP)-3alpha/CC chemokine ligand 20, in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a model of T cell-dependent inflammation. Expression in the CNS of MIP-3alpha, as determined by RT-PCR, increased in a time-dependent manner such that peak expression correlated with peak clinical disease. Similarly, levels of immunoreactive MIP-3alpha in the draining lymph nodes increased up to 10-fold 9 days postimmunization and remained elevated for up to 21 days postimmunization. The increased production of MIP-3alpha coincided with onset of clinical disease. Treatment of mice with specific neutralizing anti-MIP-3alpha Abs significantly reduced the severity of both clinical EAE and neuroinflammation by inhibiting the sensitization of lymphocytes to the specific Ag and release of lymphocytes from the draining lymph nodes. In contrast, adoptive transfer experiments indicated that MIP-3alpha was not essential for the effector phase of EAE. Together, these data demonstrate that MIP-3alpha plays a critical role in the sensitization phase of EAE.

Asunto(s)

Quimiocinas CC/fisiología , Encefalomielitis Autoinmune Experimental/inmunología , Inmunización , Proteínas Inflamatorias de Macrófagos/fisiología , Receptores de Quimiocina , Médula Espinal/inmunología , Linfocitos T/inmunología , Animales , Inhibición de Migración Celular , Movimiento Celular/inmunología , Células Cultivadas , Quimiocina CCL20 , Quimiocinas CC/antagonistas & inhibidores , Quimiocinas CC/biosíntesis , Quimiocinas CC/inmunología , Progresión de la Enfermedad , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Sueros Inmunes/farmacología , Inmunización/métodos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Proteínas Inflamatorias de Macrófagos/antagonistas & inhibidores , Proteínas Inflamatorias de Macrófagos/biosíntesis , Proteínas Inflamatorias de Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Proteína Proteolipídica de la Mielina/administración & dosificación , Proteína Proteolipídica de la Mielina/inmunología , Receptores CCR6 , Médula Espinal/metabolismo , Médula Espinal/patología , Linfocitos T/citología , Linfocitos T/patología , Regulación hacia Arriba/inmunología

RESUMEN

Eotaxin-3 (CCL26) belongs to the group of CC chemokines that attract eosinophils, basophils, and Th2 lymphocytes. Like eotaxin (CCL11) and eotaxin-2 (CCL24), eotaxin-3 mediates its activity through CCR3. Here we show that eotaxin-3 also binds to CCR2 on monocytes and CCR2-transfected cells. In contrast to monocyte chemotactic protein 1 (MCP-1; CCL2), eotaxin-3 does not trigger intracellular calcium mobilization, enzyme release, or phosphorylation of the mitogen-activated protein (MAP) kinase ERK and induces a weak chemotaxis in monocytes. Instead, eotaxin-3 inhibits MCP-1-mediated responses, thus acting as a natural antagonist for CCR2. This study also demonstrates that eotaxin-3 promotes active movement of monocytes away from a gradient of eotaxin-3 in vitro. This repellent effect is amplified when an additional gradient of MCP-1 is applied, demonstrating that the 2 mechanisms are synergistic. Eotaxin-3 effects on monocytes are largely abolished when cells are pretreated with MCP-1 or CCR2 antagonists. Like MCP-1-mediated migration, repulsion is sensitive to Bordetella pertussis toxin, indicating the involvement of Gi protein-coupled receptors. However, using transfected cells expressing CCR2 we could not detect F-actin formation or an active movement away induced by eotaxin-3, suggesting that either expression of a single receptor type is not sufficient to mediate cell repulsion or that the used transfected cell lines lack additional interaction molecules that are required for reverse migration. Eotaxin-3 was expressed by vascular endothelial cells and was essential for endothelial transmigration of eosinophils. Our data provide a mechanism by which 2 chemokine gradients that are oriented in opposite directions could cooperate in efficiently driving out monocytes from blood vessels into tissue.

Asunto(s)

Quimiocinas CC/fisiología , Quimiotaxis de Leucocito/efectos de los fármacos , Monocitos/citología , Receptores de Quimiocina/antagonistas & inhibidores , Calcio/metabolismo , Quimiocina CCL26 , Quimiocinas CC/metabolismo , Quimiocinas CC/farmacología , Humanos , Inflamación/patología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Monocitos/efectos de los fármacos , Unión Proteica , Receptores CCR2 , Transfección

RESUMEN

Cathepsin D (Cath-D) expression in human primary breast cancer has been associated with a poor prognosis. In search of a better understanding of the Cath-D substrates possibly involved in cancer invasiveness and metastasis, we investigated the potential interactions between this protease and chemokines. Here we report that purified Cath-D, as well as culture supernatants from the human breast carcinoma cell lines MCF-7 and T47D, selectively degrade macrophage inflammatory protein (MIP)-1 alpha (CCL3), MIP-1 beta (CCL4), and SLC (CCL21). Proteolysis was totally blocked by the protease inhibitor pepstatin A, and specificity of Cath-D cleavage was demonstrated using a large chemokine panel. Whereas MIP-1 alpha and MIP-1 beta degradation was rapid and complete, cleavage of SLC was slow and not complete. Mass spectrometry analysis showed that Cath-D cleaves the Leu(58) to Trp(59) bond of SLC producing two functionally inactive fragments. Analysis of Cath-D proteolysis of a series of monocyte chemoattractant protein-3/MIP-1 beta hybrids indicated that processing of MIP-1 beta might start by cleaving off amino acids located in the C-terminal domain. In situ hybridization studies revealed MIP-1 alpha, MIP-1 beta, and Cath-D gene expression mainly in the stromal compartment of breast cancers whereas SLC transcripts were found in endothelial cells of capillaries and venules within the neoplastic tissues. Cath-D production in the breast carcinoma cell lines MCF-7 and T47D, as assessed by enzyme-linked immunosorbent assay of culture supernatants and cell lysates, was not affected by stimulation with chemokines such as interleukin-8 (CXCL8), SDF-1 (CXCL12), and SLC. These data suggest that inactivation of chemokines by Cath-D possibly influences regulatory mechanisms in the tumoral extracellular microenvironment that in turn may affect the generation of the antitumoral immune response, the migration of cancer cells, or both processes.

Asunto(s)

Neoplasias de la Mama/patología , Catepsina D/metabolismo , Quimiocinas CC/metabolismo , Proteínas Inflamatorias de Macrófagos/metabolismo , Secuencia de Aminoácidos , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Neoplasias de la Mama/inmunología , Catepsina D/genética , Quimiocina CCL21 , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocinas CC/química , Quimiocinas CC/genética , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Cinética , Proteínas Inflamatorias de Macrófagos/química , Proteínas Inflamatorias de Macrófagos/genética , Datos de Secuencia Molecular , Especificidad por Sustrato , Células Tumorales Cultivadas

RESUMEN

Determining the critical structural features a ligand must possess in order to bind to its receptor is of key importance to the understanding of vital biological processes and to the rational design of small molecule therapeutics to modulate receptor function. We have developed a general strategy for determining such ligand binding motifs using low temperature NMR structures of peptides with the desired receptor binding properties. This approach has been successfully applied to determine a binding motif for the chemokine receptor CXCR4. The motif identified provides a detailed guide for the design of small molecule antagonists against CXCR4, which are much sought after to aid in the treatment of a number of conditions including human immunodeficiency virus type 1 infection and a variety of cancers.

Asunto(s)

Quimiocinas CXC/metabolismo , Fragmentos de Péptidos/farmacología , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/metabolismo , Secuencias de Aminoácidos , Unión Competitiva , Quimiocina CXCL12 , Quimiocina CXCL2 , Quimiocinas CXC/química , Humanos , Ligandos , Espectroscopía de Resonancia Magnética , Conformación Molecular , Monocinas/química , Monocinas/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Péptidos , Unión Proteica , Receptores CXCR4/química , Relación Estructura-Actividad

RESUMEN

During organogenesis, immunosurveillance, and inflammation, chemokines selectively recruit leukocytes by activating seven-transmembrane-spanning receptors. It has been suggested that an important component of this process is the formation of a haptotactic gradient by immobilization of chemokines on cell surface glycosaminoglycans (GAGs). However, this hypothesis has not been experimentally demonstrated in vivo. In the present study we investigated the effect of mutations in the GAG binding sites of three chemokines, monocyte chemoattractant protein-1/CC chemokine ligand (CCL)2, macrophage-inflammatory protein-1beta/CCL4, and RANTES/CCL5, on their ability to recruit cells in vivo. These mutant chemokines retain chemotactic activity in vitro, but they are unable to recruit cells when administered intraperitoneally. Additionally, monomeric variants, although fully active in vitro, are devoid of activity in vivo. These data demonstrate that both GAG binding and the ability to form higher-order oligomers are essential for the activity of particular chemokines in vivo, although they are not required for receptor activation in vitro. Thus, quaternary structure of chemokines and their interaction with GAGs may significantly contribute to the localization of leukocytes beyond migration patterns defined by chemokine receptor interactions.

Asunto(s)

Quimiocinas/fisiología , Glicosaminoglicanos/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Biopolímeros , Células CHO , Quimiocinas/metabolismo , Quimiotaxis de Leucocito , Cricetinae , Cartilla de ADN , Femenino , Glicosaminoglicanos/química , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Cavidad Peritoneal/citología , Proteínas Recombinantes/metabolismo

RESUMEN

The chemokine-like, secreted protein product of the U83 gene from human herpesvirus 6, here named vCCL4, was chemically synthesized to be characterized in a complete library of the 18 known human chemokine receptors expressed individually in stably transfected cell lines. vCCL4 was found to cause calcium mobilization as efficiently as the endogenous chemokine ligand CCL2 through the CCR2 receptor, whereas the virally encoded chemokine did not affect any of the other 17 human chemokine receptors tested. Mutual cross-desensitization between CCL2 and vCCL4 was demonstrated in the CCR2-transfected cells. The affinity of vCCL4 for the CCR2 receptor was 79 nm as determined in competition binding against radioactively labeled CCL2. In the murine pre-B lymphocyte cell line L1.2 stably transfected with the CCR2 receptor, vCCL4 acted as a relatively low potency but highly efficacious chemoattractant being equally or more efficacious in causing cell migration than CCL2 and CCL7 and considerably more efficacious than CCL8 and CCL13. It is concluded that human herpesvirus 6 encodes a highly selective and efficacious CCR2 agonist, which will attract CCR2 expressing cells, for example macrophages and monocytes, conceivably for the virus to infect and to establish latency in. It is suggested that vCCL4 during reactivation of the virus in for example monocyte-derived microglia could perhaps be involved in the pathogenesis of the CCR2-dependent disease, multiple sclerosis.

Asunto(s)

Quimiocinas/genética , Herpesvirus Humano 6/genética , Receptores de Quimiocina/agonistas , Proteínas Virales/genética , Secuencia de Aminoácidos , Animales , Células CHO , Células COS , Cricetinae , Genes Virales , Datos de Secuencia Molecular , Receptores CCR2 , Receptores de Quimiocina/química , Receptores de Quimiocina/metabolismo , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido

RESUMEN

I-TAC, IP10, and Mig are interferon-gamma inducible CXC chemokines that share the same G-protein-coupled receptor CXCR3, which is preferentially expressed on Th1 lymphocytes. We have explored the structure-function relationship of the CXCR3 ligands, in particular of I-TAC, which has highest affinity for CXCR3 and is the most potent agonist. A potent antagonist for CXCR3 was obtained by NH(2)-terminal truncation of I-TAC. I-TAC (4-73), which lacks the first three residues, has no agonistic activity but competes for the binding of I-TAC to CXCR3-bearing cells and inhibits migration and Ca(2+) changes in such cells in response to stimulation with I-TAC, IP10, and Mig. It does also not induce internalization of CXCR3, which is in support of the lack of agonistic effects. Hybrid chemokines between I-TAC and IP10 were used to identify regions responsible for the higher activity of I-TAC. I-TAC-like IP10 analogs are obtained by substituting the NH(2) terminus (residues 1-8) or N-loop region (residues 12-17) of IP10 with those of I-TAC, suggesting that the differences in function of the CXCR3 ligands can be assigned to distinct regions and that these regions are interchangeable. Structure-activity studies with Mig showed that the extended basic COOH-terminal region, which is not present in I-TAC and IP10, is important for binding and activity.

Asunto(s)

Quimiocinas CXC/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Receptores de Quimiocina/metabolismo , Secuencia de Aminoácidos , Calcio/metabolismo , Línea Celular , Células Cultivadas , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocina CXCL9 , Quimiocinas CXC/química , Quimiocinas CXC/genética , Quimiotaxis/fisiología , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Ligandos , Datos de Secuencia Molecular , Unión Proteica , Ensayo de Unión Radioligante , Receptores CXCR3 , Receptores de Quimiocina/antagonistas & inhibidores , Receptores de Quimiocina/química , Alineación de Secuencia , Relación Estructura-Actividad

RESUMEN

Increasingly it is being recognized that matrix metalloproteinases (MMPs) are important processing enzymes that regulate cellular behaviour and immune cell function by selective proteolysis of cell surface receptors and adhesion molecules, cytokines and growth factors. These functions will likely prove to be as important in vivo as the proposed roles of MMPs in pathological matrix degradation. To screen for new protease substrates we have reported a novel 'exosite scanning' strategy that utilizes protease substrate-binding exosite domains as yeast two-hybrid baits. We discovered that the chemokine monocyte chemoattractant protein-3 (MCP-3) binds the hemopexin C domain of gelatinase A (MMP-2) leading to its efficient cleavage, converting an agonist to a potent receptor antagonist. We have now found that other MMPs cleave MCP-1, MCP-2, MCP-3, MCP-4, SDF-lalpha and SDF-1beta indicating that the intersection between the chemokine and MMP families is broad with important implications for the control of inflammatory and immune processes. Use of engineered substrates with altered exosite binding affinities further revealed the power of exosites in dictating proteolytic specificity - either directing cleavage of non-preferred sites or in other cases virtually eliminating proteolysis of readily accessible scissile bonds. Hence, bioinformatic searches for protease substrates based on scissile bond preference will only reveal a subset of substrates unless the influence of exosites is considered.

Asunto(s)

Quimiocinas/metabolismo , Metaloendopeptidasas/metabolismo , Técnicas del Sistema de Dos Híbridos , Animales , Matriz Extracelular/metabolismo , Humanos , Metaloendopeptidasas/química , Especificidad por Sustrato

RESUMEN

Monocyte chemoattractant protein (MCP)-3 is inactivated upon cleavage by the matrix metalloproteinase (MMP) gelatinase A (MMP-2). We investigated the susceptibility to proteolytic processing of the 4 human MCPs by 8 recombinant MMPs to determine whether MCP-3 is an isolated example or represents a general susceptibility of chemokines to proteolytic inactivation by these important inflammatory proteases. In addition to MMP-2, MCP-3 is efficiently cleaved by membrane type 1 (MT1)-MMP, the cellular activator of MMP-2, and by collagenase-1 and collagenase-3 (MMP-1, MMP-13) and stromelysin-1 (MMP-3). Specificity was shown by absence of cleavage by matrilysin (MMP-7) and the leukocytic MMPs neutrophil collagenase (MMP-8) and gelatinase B (MMP-9). The closely related chemokines MCP-1, MCP-2, and MCP-4 were not cleaved by MMP-2 or MT1-MMP, but were cleaved by MMP-1 and MMP-3 with varying efficiency. MCPs were typically cleaved between residues 4 and 5, but MCP-4 was further processed at Val7-Pro8. Synthetic MCP analogs corresponding to the MMP-cleaved forms bound CC chemokine receptor (CCR)-2 and CCR-3, but lacked chemoattractant activity in pre-B cells transfected with CCR-2 and CCR-3 or in THP-1 monocytic cells, a transformed leukemic cell line. Moreover, the truncated products of MCP-2 and MCP-4, like MCP-3, were potent antagonists of their cognate CC chemokine receptors in transwell cell migration assays in vitro. When they were injected 24 hours after the initiation of carrageenan-induced inflammation in rat paws, their in vivo antagonist activities were revealed by a greater than 66% reduction in inflammatory edema progression after 12 hours. We propose that MMPs have an important role in modulating inflammatory and immune responses by processing chemokines in wound healing and in disease.

Asunto(s)

Antiinflamatorios , Citocinas , Metaloproteinasas de la Matriz/metabolismo , Proteínas Quimioatrayentes de Monocitos/metabolismo , Receptores de Quimiocina/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Linfocitos B/metabolismo , Células CHO , Carragenina , Quimiocina CCL2/metabolismo , Quimiocina CCL7 , Quimiocina CCL8 , Colagenasas/metabolismo , Cricetinae , Edema/inducido químicamente , Edema/tratamiento farmacológico , Activación Enzimática , Femenino , Humanos , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Masculino , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 13 de la Matriz , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/metabolismo , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Ratas , Ratas Wistar , Receptores de Quimiocina/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Transfección

RESUMEN

The structure of IP-10 was solved by NMR spectroscopy and represents the first structure from the class of agonists toward the receptor CXCR3. CXCR3 binding chemokines are unique in their ability to bind receptors from both the CC and CXC classes of chemokine receptors. An unusual structural feature of IP-10 was identified that may provide the basis for the ability of IP-10 to bind both CXCR3 and CCR3. The surface of IP-10 that interacts with the N-terminus of CXCR3 was defined by monitoring changes in the NMR spectrum of IP-10 upon addition of a CXCR3 N-terminal peptide. These studies indicated that the interaction involves a hydrophobic cleft, formed by the N-loop and 40s-loop region of IP-10, similar to the interaction surface observed for other chemokines such as IL-8. An additional region of interaction was observed that consists of a hydrophobic cleft formed by the N-terminus of IP-10 and 30s-loop of IP-10.

Asunto(s)

Quimiocinas CXC/química , Quimiocinas CXC/metabolismo , Receptores de Quimiocina/química , Receptores de Quimiocina/metabolismo , Quimiocina CXCL10 , Dimerización , Humanos , Modelos Moleculares , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Protones , Receptores CXCR3