RESUMEN
Adhesive interactions play an important role in inflammation by promoting leukocyte attachment and extravasation from the vasculature into the peripheral tissues. However, the importance of adhesion molecules within the extracellular matrix-rich environment of peripheral tissues, in which cells must migrate and be activated, has not been well explored. We investigated the role of the major collagen-binding integrins, alpha1beta1 and alpha2beta1, in several in vivo models of inflammation. mAb's against murine alpha1 and alpha2 were found to significantly inhibit effector phase inflammatory responses in animal models of delayed-type hypersensitivity (DTH), contact hypersensitivity (CHS), and arthritis. Mice that were alpha1-deficient also showed decreased inflammatory responses in the CHS and arthritis models when compared with wild-type mice. Decreased leukocyte infiltration and edema formation accompanied inhibition of antigen-specific models of inflammation, as nonspecific inflammation induced by croton oil was not inhibited. This study demonstrates the importance in vivo of alpha1beta1 and alpha2beta1, the collagen-binding integrins, in inflammatory diseases. The study also extends the role of integrins in inflammation beyond leukocyte attachment and extravasation at the vascular endothelial interface, revealing the extracellular matrix environment of peripheral tissues as a new point of intervention for adhesion-based therapies.
Asunto(s)
Artritis/prevención & control , Adhesión Celular/fisiología , Colágeno/metabolismo , Dermatitis Alérgica por Contacto/prevención & control , Hipersensibilidad Tardía/prevención & control , Integrinas/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Artritis/inmunología , Artritis/patología , Colágeno/toxicidad , Dermatitis Alérgica por Contacto/inmunología , Dermatitis Alérgica por Contacto/patología , Dermatitis Irritante/inmunología , Dermatitis Irritante/patología , Dermatitis Irritante/prevención & control , Edema/etiología , Edema/prevención & control , Femenino , Hipersensibilidad Tardía/inmunología , Hipersensibilidad Tardía/patología , Integrina alfa1beta1 , Integrinas/inmunología , Leucocitos/patología , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptores de ColágenoRESUMEN
Central to immune and inflammatory responses are the integrin-mediated adhesive interactions of cells with their extracellular matrix (ECM)-rich environment. Using a comprehensive and quantitative mRNA profiling technique, we analyzed the effect of ECM-induced attachment on monocyte gene expression, its regulation by growth factors, and the integrin specificity of this event. Adhesion of cells to fibronectin resulted in increased expression of a large number of genes, which was strongly potentiated by the presence of growth factors. Adhesion activated both the NF-kappaB and Jak/STAT pathways of gene transcription and increased expression of genes involved in inflammatory and immune responses, revealing the importance of ECM-integrin interactions in these processes.
Asunto(s)
Matriz Extracelular/metabolismo , Integrinas/metabolismo , Monocitos/metabolismo , Adhesión Celular/fisiología , Línea Celular , Proteínas de Unión al ADN/metabolismo , Fibronectinas/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Sustancias de Crecimiento/metabolismo , Humanos , Janus Quinasa 1 , FN-kappa B/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción STAT1 , Transducción de Señal , Transactivadores/metabolismoRESUMEN
During development, sonic hedgehog functions as a morphogen in both a short-range contact-dependent and in a long-range diffusable mode. Here, we show using a panel of sonic hedgehog variants that regions near the N terminus of the protein play a critical role in modulating these functions. In the hedgehog responsive cell line C3H10T1/2, we discovered that not only were some N-terminally truncated variants inactive at eliciting a hedgehog-dependent response, but they competed with the wild-type protein for function and therefore served as functional antagonists. These variants were indistinguishable from wild-type sonic hedgehog in their ability to bind the receptor patched-1, but failed to induce the hedgehog-responsive markers, Gli-1 and Ptc-1, and failed to promote hedgehog-dependent differentiation of the cell line. They also failed to support the adhesion of C3H10T1/2 cells to hedgehog-coated plates under conditions where wild-type sonic hedgehog supported binding. Structure-activity data indicated that the N-terminal cysteine plays a key regulatory role in modulating hedgehog activity. The ability to dissect patched-1 binding from signaling events in C3H10T1/2 cells suggests the presence of unidentified factors that contribute to hedgehog responses.
Asunto(s)
Fosfatasa Alcalina/genética , Proteínas/química , Proteínas/metabolismo , Transactivadores , Fosfatasa Alcalina/biosíntesis , Animales , Sitios de Unión , Adhesión Celular , Línea Celular , Movimiento Celular , Embrión de Pollo , Clonación Molecular , Inducción Embrionaria , Inducción Enzimática , Escherichia coli , Proteínas Hedgehog , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Ratones , Ratones Transgénicos , Sistema Nervioso/citología , Sistema Nervioso/embriología , Proteínas Oncogénicas/metabolismo , Técnicas de Cultivo de Órganos , Receptores Patched , Receptor Patched-1 , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Pichia , Proteínas/antagonistas & inhibidores , Proteínas/genética , Receptores de Superficie Celular , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , beta-Galactosidasa/genéticaRESUMEN
Recent structural and functional analyses of alpha integrin subunit I domains implicate a region in cation and ligand binding referred to as the metal ion-dependent adhesion site (MIDAS). Although the molecular interactions between Mn2+ and Mg2+ and the MIDAS region have been defined by crystallographic analyses, the role of cation in I domain function is not well understood. Recombinant alpha 1 beta 1 integrin I domain (alpha1-I domain) binds collagen in a cation-dependent manner. We have generated and characterized a panel of antibodies directed against the alpha1-I domain, and selected one (AJH10) that blocks alpha 1 beta 1 integrin function for further study. The epitope of AJH10 was localized within the loop between the alpha 3 and alpha 4 helices which contributes one of the metal coordination sites of the MIDAS structure. Kinetic analyses of antibody binding to the I domain demonstrate that divalent cation is required to stabilize the epitope. Denaturation experiments demonstrate that cation has a dramatic effect on the stabilization of the I domain structure. Mn2+ shifts the point at which the I domain denatures from 3.4 to 6.3 M urea in the presence of the denaturant, and from 49.5 to 58.6 degrees C following thermal denaturation. The structural stability provided to the alpha1-I domain by divalent cations may contribute to augmented ligand binding that occurs in the presence of these cations.
Asunto(s)
Cationes Bivalentes/química , Integrinas/química , Magnesio/fisiología , Manganeso/fisiología , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Colágeno/metabolismo , Epítopos/química , Epítopos/metabolismo , Femenino , Humanos , Integrina alfa1beta1 , Integrinas/antagonistas & inhibidores , Integrinas/genética , Integrinas/inmunología , Magnesio/química , Manganeso/química , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Unión Proteica/genética , Unión Proteica/inmunología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Integrins are a major class of cell surface receptors involved in cell-cell and cell-matrix adhesion and communication. Ha2/11 is a function-blocking anti-rat beta 1 integrin hamster IgM that should be a useful reagent for understanding beta 1 integrin function. We demonstrate that Ha2/11 cross reacts with human, Xenopus, and Drosophila beta 1 integrins, and use phage display to map the epitope for Ha2/11 to residues within the sequence LRSGEPQTF which lies 18 amino acids proximal to the putative I domain in beta 1 integrins. Monoclonal antibody mapping experiments, mutational analyses, and direct binding assays have implicated integrin I domains in both cation and ligand binding. Our data therefore suggest that Ha2/11 blocks beta 1 integrin function by interfering with I domain-mediated ligand binding.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Colifagos/genética , Epítopos/química , Inmunoglobulina M/farmacología , Integrina beta1/inmunología , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Reacciones Antígeno-Anticuerpo , Línea Celular , Pollos/inmunología , Secuencia de Consenso , Cricetinae , Reacciones Cruzadas , Drosophila melanogaster/citología , Drosophila melanogaster/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos/metabolismo , Citometría de Flujo , Humanos , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Técnicas de Inmunoadsorción , Riñón/citología , Ligandos , Ratones , Músculo Liso Vascular/citología , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Unión Proteica , Ratas , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Xenopus laevis/inmunologíaRESUMEN
Many cell-surface and extracellular matrix proteins contain multiple modular domains known as fibronectin type III (FNIII) repeats. Cells adhere to the extracellular matrix proteins fibronectin and tenascin in part by the interaction of certain integrins with the Arg-Gly-Asp (RGD) sequence, displayed on specific FNIII repeats. We have found that, after experimental activation of beta1 integrins, a number of cell types adhere and spread on FNIII repeats lacking RGD, derived from extracellular matrix proteins and cytokine receptors. Interaction between individual FNIII domains and beta1 integrins mediates focal adhesion kinase phosphorylation and subsequent stress fiber and focal contact formation. These data suggest that many FNIII-containing proteins may bind and signal through activated beta1 integrins, dramatically expanding the potential for integrin-dependent intercellular and cell-matrix communication.
Asunto(s)
Adhesión Celular/fisiología , Fibronectinas/química , Integrina beta1/fisiología , Oligopéptidos/fisiología , Transducción de Señal/fisiología , Animales , Sitios de Unión , Calcio/metabolismo , Moléculas de Adhesión Celular/metabolismo , Comunicación Celular/fisiología , Proteínas de la Matriz Extracelular/química , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Manganeso/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Ratas , Receptores de Citocinas/químicaRESUMEN
Remodeling of the extracellular matrix by activated mesenchymal cells (myofibroblasts) is a critical aspect of wound repair in all adult organs. Collagen-dependent gel contraction, a process requiring integrin function, is an established in vitro assay thought to mimic in vivo matrix remodeling. Numerous data have implicated the alpha2beta1 integrin in various cell types as the primary collagen receptor responsible for collagen gel contraction. However, evidence from the literature suggests that the major collagen binding integrin expressed on mesenchymally derived cells in situ is the alpha1beta1 integrin, not the alpha2beta1 integrin. In this report, we use a rat vascular injury model to illustrate that the alpha1beta1 integrin is the major collagen receptor expressed on vascular smooth muscle cells after injury. Using two smooth muscle cell lines, expressing either the alpha1beta1 integrin alone or both the alpha1beta1 and alpha2beta1 integrins, along with Chinese hamster ovary cells transfected with the alpha1 integrin, we demonstrate that alpha1beta1 supports not only collagen-dependent adhesion and migration, but also gel contraction. These data suggest that in vivo the alpha1beta1 integrin is a critical collagen receptor on mesenchymally derived cells potentially involved in matrix remodeling after injury.
Asunto(s)
Arteria Carótida Común/fisiología , Colágeno/metabolismo , Integrinas/biosíntesis , Músculo Liso Vascular/fisiología , Túnica Íntima/fisiología , Cicatrización de Heridas , Animales , Antígenos CD/biosíntesis , Aorta/lesiones , Aorta/fisiología , Células CHO , Traumatismos de las Arterias Carótidas , Adhesión Celular , Línea Celular , Movimiento Celular , Cricetinae , Matriz Extracelular/fisiología , Humanos , Inmunohistoquímica , Hibridación in Situ , Integrina alfa1 , Integrina alfa1beta1 , Masculino , Arteria Pulmonar/fisiología , Ratas , Ratas Sprague-Dawley , TransfecciónRESUMEN
Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the integrin very late antigen-4 (VLA4). The VCAM1/VLA4 interaction mediates both adhesion and signal transduction and is thought to play an important role in inflammatory and immune responses in vivo. The major form of human VCAM1 contains seven extracellular Ig-like domains, with domain 1 designated as the most N-terminal. We have examined the relationship between human VCAM1 structure and function using a combination of domain truncation mutants and proteolytic fragmentation of recombinant soluble VCAM1. We have characterized two regions of VCAM1, localized to domains 4 and 5, which are highly sensitive to proteolytic cleavage, localized the epitope of the blocking monoclonal antibody 4B9 to domain 1, and found that domains 1-3 are sufficient for both its adhesive function and its ability to initiate T cell activation.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Adhesión Celular , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/fisiología , Línea Celular , Humanos , Activación de Linfocitos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Fragmentos de Péptidos/aislamiento & purificación , Receptores de Antígeno muy Tardío/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal , Linfocitos T/inmunología , Transfección , Molécula 1 de Adhesión Celular VascularRESUMEN
The infiltration of the synovial membrane (SM) by mononuclear cells, mostly T cells, is a typical histopathological feature associated with rheumatoid arthritis (RA). The entry of T lymphocytes into the SM is believed to be mediated by a number of molecules in the endothelium that are induced in response to a series of inflammatory mediators. In this study, we have investigated the adhesion of synovial T cells from RA patients to two endothelial ligands: endothelial-leukocyte adhesion molecule-1 (ELAM-1), the only selectin known to function as a vascular addressin for T cells, and vascular cell adhesion molecule-1 (VCAM-1), the cellular ligand of VLA-4. Our results clearly demonstrate that synovial T cells isolated from both SM and synovial fluid (SF), bearing an activated and memory phenotype, displayed an enhanced capacity to interact with these two endothelial molecules as compared with T cells from peripheral blood (PB) either of the same RA patients or healthy donors. A further enhancement of VLA-4-mediated T cell binding to VCAM-1 and fibronectin could be observed when already in vivo-activated synovial T cells were stimulated in vitro with phorbol esters, suggesting the existence of several cellular affinity levels for both very late activation-4 (VLA-4) ligands. Moreover, both PB and synovial T cells from RA patients exhibited strong proliferative responses when they were cultured with either fibronectin or VCAM-1 in combination with submitogenic doses of anti-CD3 mAb. This increased endothelial binding ability of synovial T lymphocytes together with their proliferation in response to the interaction with VCAM-1 and fibronectin may represent important mechanisms in the regulation of T cell penetration and persistence in the chronically inflamed SM of RA.
Asunto(s)
Artritis Reumatoide/inmunología , Moléculas de Adhesión Celular/metabolismo , Adhesión Celular , Linfocitos T/citología , Antígenos CD/análisis , Antígenos de Diferenciación de Linfocitos T/análisis , Artritis Reumatoide/patología , Colágeno/metabolismo , Selectina E , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Fibronectinas/metabolismo , Antígenos de Histocompatibilidad/análisis , Humanos , Lectinas Tipo C , Antígenos Comunes de Leucocito , Activación de Linfocitos , Receptores de Antígeno muy Tardío/análisis , Líquido Sinovial/inmunología , Membrana Sinovial/inmunología , Membrana Sinovial/patología , Molécula 1 de Adhesión Celular VascularRESUMEN
Vascular cell adhesion molecule, VCAM-1, is an adhesion molecule expressed on activated endothelium thought to play a role in leukocyte migration to sites of inflammation. VCAM-1 adheres to leukocytes through the VLA-4 integrin. Recombinant soluble VCAM-1 (rsVCAM) and anti-CD3 mAb OKT3 were utilized to address the role of the VCAM-1/VLA-4 pathway in antigen-dependent T cell activation. Monocyte-depleted T cells proliferated upon exposure to co-immobilized OKT3 and rsVCAM but to neither alone. In contrast, an anti-VLA-4 mAb HP1/2 failed to co-activate with OKT3, despite the fact that both rsVCAM and HP1/2 support T cell adhesion comparably. These data indicate that adhesive function is not sufficient for co-stimulatory activity. They also reveal that VCAM-1 may play a role in regulating T cell immune responses as well as migration in vivo.
Asunto(s)
Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos T/fisiología , Moléculas de Adhesión Celular/fisiología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/fisiología , Receptores de Antígeno muy Tardío/fisiología , Linfocitos T/fisiología , Complejo CD3 , Adhesión Celular , Humanos , Técnicas In Vitro , Proteínas Recombinantes/metabolismo , Transducción de Señal , Molécula 1 de Adhesión Celular VascularRESUMEN
Adherence of human eosinophils to cytokine-stimulated endothelial cells, which was only partially due to CD18-dependent pathways, was also mediated by binding to endothelial leukocyte adhesion molecule 1 (ELAM-1) and vascular cell adhesion molecule 1 (VCAM-1). Eosinophils bound specifically to both recombinant soluble ELAM-1 and recombinant soluble VCAM-1. Eosinophil binding to recombinant soluble VCAM-1 and to transfected CHO cells expressing VCAM-1 was inhibited with anti-VCAM-1 (4B9) and anti-very late activation antigen 4 (anti-VLA-4; HP1/2 or HP2/1) monoclonal antibodies. Eosinophils, but not neutrophils, expressed VLA-4 detected by cytofluorography. Eosinophil adherence to tumor necrosis factor alpha-stimulated human umbilical vein endothelial cells was partially blocked by monoclonal antibodies against ELAM-1 (BB11) and VCAM-1 (4B9) and against VLA-4 (HP2/1). Thus, while both eosinophils and neutrophils can bind to activated endothelial cells by adherence to ICAM-1 and ELAM-1, only eosinophils expressed VLA-4 and adhered to VCAM-1 on activated endothelial cells. Eosinophil adherence to VCAM-1 might provide a mechanism contributing to the selective recruitment of eosinophils into tissue sites of inflammation.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Adhesión Celular , Endotelio Vascular/fisiología , Eosinófilos/fisiología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Selectina E , Humanos , Neutrófilos/fisiología , Unión Proteica , Factor de Necrosis Tumoral alfa/farmacología , Venas Umbilicales , Molécula 1 de Adhesión Celular VascularRESUMEN
Vascular cell adhesion molecule 1 (VCAM1) is a leukocyte adhesion molecule induced on human endothelium in vitro and in vivo by inflammatory stimuli. A truncated cDNA for VCAM1 was constructed, stably expressed in Chinese Hamster Ovary (CHO) cells, and the secreted recombinant soluble form of VCAM1 (rsVCAM1) purified to homogeneity by immunoaffinity chromatography. Immobilized rsVCAM1 is a functional adhesion protein, and selectively binds only VLA4-expressing cells, including human B and T lymphocytes, NK cells, and certain lymphoblastoid cell lines. T cell subset analyses indicate preferential binding of CD8+ memory cells. rsVCAM1 should prove valuable for the further study of the role of VCAM1 during inflammatory and immune responses in vivo.
Asunto(s)
Moléculas de Adhesión Celular/genética , Adhesión Celular/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Moléculas de Adhesión Celular/aislamiento & purificación , Moléculas de Adhesión Celular/farmacología , Línea Celular , Membrana Celular/fisiología , Codón , Citoplasma/fisiología , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/fisiología , Transfección , Molécula 1 de Adhesión Celular VascularRESUMEN
Endothelial leukocyte adhesion molecule 1 (ELAM1) is a leukocyte adhesion molecule induced on human venular endothelium in vitro and in vivo by inflammatory stimuli. A truncated cDNA for ELAM1 has been constructed, stably expressed in Chinese hamster ovary cells, and the secreted recombinant soluble form of ELAM1 (rsELAM1) purified to homogeneity by immunoaffinity chromatography. rsELAM1, when immobilized on plastic, is fully functional as an adhesion protein, and selectively binds only cells known to bind cell-surface ELAM1 expressed on human endothelial cells, including the myelomonocytic cell line HL60 and the colon carcinoma cell line HT29. Immobilized rsELAM1 also binds human PMN, monocytes, NK cells, and T cells. T cell subset analyses indicate preferential binding of CD4+ T memory cells. However, rsELAM1 is only a weak inhibitor of ELAM1-mediated adhesion. rsELAM1 should prove valuable for the further study of the role of ELAM1 expressed on the vascular wall during the inflammatory response.
Asunto(s)
Moléculas de Adhesión Celular/genética , Secuencia de Aminoácidos , Secuencia de Bases , Unión Competitiva , Adhesión Celular , Moléculas de Adhesión Celular/química , Línea Celular , Clonación Molecular , Selectina E , Humanos , Leucocitos/citología , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Proteínas Recombinantes , SolubilidadRESUMEN
Two classes of adhesion molecules have well-defined roles in the attachment of unstimulated polymorphonuclear leukocytes (PMN) to cytokine-treated endothelial cells. Endothelial-leukocyte adhesion molecule 1 (ELAM-1) on endothelial cells interacts with specific carbohydrate residues on the PMN, and the leukocyte integrins (CD18 antigens) on PMN interact with intracellular adhesion molecule 1 and other structures on endothelium. Here we show that these two classes of molecules can act sequentially in an "adhesion cascade". Interaction of PMN with ELAM-1-bearing endothelial cells causes PMN to express enhanced adhesive activity of the integrin CR3 (CD11b/CD18). Expression of ELAM-1 on the cytokine-treated endothelium appears both necessary and sufficient for the stimulation of CR3 activity since blockade of ELAM-1 with mAbs prevents the activation of CR3 by cytokine-treated endothelium, and immobilized recombinant ELAM-1 activates CR3. The ability to activate CR3 is shared by chemattractants, suggesting that ELAM-1 may serve as a "tethered chemattractant." This hypothesis is strengthened by the observation that recombinant soluble ELAM-1 directs movement of PMN in chemotaxis chambers. These results suggest a mechanism by which multiple adhesive molecules may function together in diapedesis. ELAM-1 serves both as an adhesin and as a trigger that recruits the participation of additional adhesion molecules. Our results also suggest that ligands for adhesion molecules may also be "receptors" capable of generating intracellular signals.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Adhesión Celular , Antígeno de Macrófago-1/fisiología , Neutrófilos/citología , Anticuerpos Monoclonales , Factores Quimiotácticos/fisiología , Quimiotaxis de Leucocito , Selectina E , Endotelio Vascular/citología , Humanos , Técnicas In Vitro , Integrinas/fisiología , Interleucina-1/farmacología , Interleucina-8/farmacología , Lipopolisacáridos/farmacología , Proteínas Recombinantes , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Vascular cell adhesion molecule-1 (VCAM1) of the Ig superfamily is induced by the inflammatory cytokines interleukin-1 and tumor necrosis factor on human umbilical vein endothelial cells (HUVECs). It binds to mononuclear leukocytes via the integrin VLA-4. We have cloned and expressed a cDNA encoding a new form of human VCAM1 containing an additional Ig homologous domain inserted between the third and fourth domains of the original six-domain protein. Characterization of mRNA from HUVECs from three individuals at various time points after induction by tumor necrosis factor indicates that both the long and short VCAM1 mRNAs are made by all three individuals, with the long form predominating quantitatively. Immunoprecipitation of VCAM1 protein from cos7 cells transfected with each cDNA and from cultured endothelial cells followed by deglycosylation suggests that the long form is the major form found on endothelium. The two forms may result from alternate splicing of a precursor mRNA. Both forms support adhesion of VLA-4-expressing cell lines.
Asunto(s)
Moléculas de Adhesión Celular/genética , Variación Genética , Secuencia de Aminoácidos , Secuencia de Bases , Moléculas de Adhesión Celular/fisiología , Células Cultivadas , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Endotelio Vascular/fisiología , Femenino , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Embarazo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Homología de Secuencia de Ácido Nucleico , Transfección , Venas Umbilicales , Molécula 1 de Adhesión Celular VascularRESUMEN
The LECCAMs are a family of cell adhesion molecules implicated in certain inflammatory processes. ELAM-1, a LECCAM found on the surface of activated endothelial cells, can mediate adhesion of neutrophils, monocytes, and certain cell lines to endothelial cells in vitro. No ligand for any LECCAM has yet been fully characterized. Here we report the cloning of a cDNA, ELFT (ELAM-1 ligand fucosyltransferase), that can confer ELAM-1 binding activity when transfected into nonbinding cell lines. ELFT encodes a 46 kd protein that has alpha(1,3)fucosyltransferase activity, suggesting that a fucosylated carbohydrate structure is an essential component of the ELAM-1 ligand. Furthermore, ELFT is expressed specifically in cell types that bind to ELAM-1, suggesting that this enzyme is an important regulator of inflammatory events in vivo.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Fucosiltransferasas/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Antígenos de Superficie/genética , Secuencia de Bases , Adhesión Celular , Línea Celular , ADN/genética , ADN/aislamiento & purificación , Selectina E , Citometría de Flujo , Biblioteca de Genes , Humanos , Antígeno Lewis X , Ligandos , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia MolecularRESUMEN
The expression and function of a new cytokine-induced endothelial cell adhesion protein, vascular cell adhesion molecule-1 (VCAM-1), was characterized in vitro by using a monoclonal antibody, MoAb 4B9, which recognizes a functional epitope on this protein. As determined by enzyme-linked immunosorbent assay and radioimmunoprecipitation of metabolically labeled cells, VCAM-1 was minimally expressed on unstimulated human umbilical vein endothelium (HUVE), but was rapidly induced by recombinant human tumor necrosis factor-alpha (rhTNF-alpha), rh interleukin-1, and lipopolysaccharide. In contrast to intercellular adhesion molecule-1, VCAM-1 was not induced on dermal fibroblasts or arterial smooth muscle cells after stimulation with rhTNF, or on keratinocytes after stimulation with rh interferon-gamma. MoAb 4B9 significantly inhibited the adherence of peripheral blood lymphocytes (PBL) and lymphocytic cell lines, but not neutrophils, to rhTNF-activated HUVE. The inhibitory effect of MoAb 4B9 on PBL adherence to HUVE was additive to that produced by the CD18 MoAb 60.3. These results show that VCAM-1 mediates a CD18-independent pathway of peripheral blood lymphocyte adherence to cytokine-stimulated HUVE. We propose that lymphocyte binding to VCAM-1, induced on endothelium by cytokines, may be an important component of lymphocyte emigration at sites of inflammation or immune reaction.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Adhesión Celular/efectos de los fármacos , Endotelio Vascular/fisiología , Linfocitos/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Anticuerpos Monoclonales , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/sangre , Moléculas de Adhesión Celular/genética , Línea Celular , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos , Humanos , Molécula 1 de Adhesión Intercelular , Linfocitos/efectos de los fármacos , Plásmidos , Proteínas Recombinantes/farmacología , Transfección , Molécula 1 de Adhesión Celular VascularRESUMEN
ELAM1 is a leukocyte adhesion molecule induced on human umbilical vein endothelial cells (HUVECs) by inflammatory cytokines. Balb/C mice were immunized with COS cells transiently expressing cell-surface ELAM1 after transfection with ELAM1 cDNA. After fusion, ELAM1-specific monoclonal antibodies (Mabs) were identified by selective adhesion to ELAM1-expressing, but not control, CHO cells, and to cytokine-treated but not untreated HUVECs. One Mab, designated BB11, binds to and immunoprecipitates ELAM1 expressed on HUVECs, COS and CHO cells. BB11 blocks the interaction of ELAM1 with human PMN, the human myelomonocytic cell line HL60, and the human colon carcinoma line HT29.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Moléculas de Adhesión Celular/inmunología , Endotelio Vascular/inmunología , Adhesión Celular , Selectina E , Endotelio Vascular/citología , Humanos , Técnicas In Vitro , Glicoproteínas de Membrana/inmunología , Neutrófilos/citología , Proteínas Recombinantes/inmunología , Transfección , Células Tumorales CultivadasRESUMEN
A cDNA for endothelial leukocyte adhesion molecule 1 (ELAM-1) was isolated by transient expression in COS-7 cells of a subtracted cDNA library from cytokine-treated human umbilical vein endothelial cells (HUVECs), with selection of ELAM-1-expressing clones by adhesion of transfected cells to the human promyelocytic cell line HL-60. This cloning method requires neither antibody nor purified ligand. ELAM-1-expressing COS cells bind the promyelocytic cell line HL-60 by a Ca2(+)-dependent but temperature-independent mechanism. Although ELAM-1 is homologous to mammalian lectins, its interaction with HL-60 cells is not inhibited by simple carbohydrate structures. ELAM-1-expressing COS cells also bind human neutrophils and the human colon carcinoma cell line HT-29, but not the B-cell line Ramos. However, Ramos cells adhere to cytokine-treated HUVECs but not control HUVECs, confirming the existence of other inducible adhesion molecules. In addition, the binding of HL-60 cells or neutrophils to ELAM-1-expressing COS cells is not inhibited by a monoclonal antibody (60.3) directed to an inhibitory epitope on CD18, indicating that the ELAM-1 ligand, although uncharacterized, is not a member of the CD11/CD18 family.
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Moléculas de Adhesión Celular/genética , ADN/genética , Glicoproteínas de Membrana/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Adhesión Celular , Moléculas de Adhesión Celular/biosíntesis , Línea Celular , Células Cultivadas , Clonación Molecular , Selectina E , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Expresión Génica , Biblioteca de Genes , Humanos , Interleucina-1/farmacología , Datos de Secuencia Molecular , Proteínas Recombinantes/farmacología , TransfecciónRESUMEN
The distribution of hyaluronate-binding activity was determined in the soluble and membrane fractions derived from adult mouse brain by sonication in low-ionic-strength buffer. Approximately 60% of the total activity was recovered in the soluble fraction and 33% in membrane fractions. In both cases, the hyaluronate-binding activities were found to be of high affinity (KD = 10(-9) M), specific for hyaluronate, and glycoprotein in nature. Most of the hyaluronate-binding activity from the soluble fraction chromatographed in the void volume of Sepharose CL-4B and CL-6B. Approximately 50% of this activity was highly negatively charged, eluting from diethylaminoethyl (DEAE)-cellulose in 0.5 M NaCl, and contained chondroitin sulfate chains. This latter material also reacted with antibodies raised against cartilage link protein and the core protein of cartilage proteoglycan. Thus, the binding and physical characteristics of this hyaluronate-binding activity are consistent with those of a chondroitin sulfate proteoglycan aggregate similar to that found in cartilage. A 500-fold purification of this proteoglycan-like, hyaluronate-binding material was achieved by wheat germ agglutinin affinity chromatography, molecular sieve chromatography on Sepharose CL-6B, and ion exchange chromatography on DEAE-cellulose. Another class of hyaluronate-binding material (25-50% of that recovered) eluted from DEAE with 0.24 M NaCl; this material had the properties of a complex glycoprotein, did not contain chondroitin sulfate, and did not react with the antibodies against cartilage link protein and proteoglycan. Thus, adult mouse brain contains at least three different forms of hyaluronate-binding macromolecules. Two of these have properties similar to the link protein and proteoglycan of cartilage proteoglycan aggregates; the third is distinguishable from these entities.