RESUMEN
Pannexin1 (Panx1) forms ATP channels that play a critical role in the immune response by reinforcing purinergic signal amplification in the immune synapse. Platelets express Panx1 and given the importance of ATP release in platelets, we investigated Panx1 function in platelet aggregation and the potential impact of genetic polymorphisms on Panx1 channels. We show here that Panx1 forms ATP release channels in human platelets and that inhibiting Panx1 channel function with probenecid, mefloquine or specific (10)Panx1 peptides reduces collagen-induced platelet aggregation but not the response induced by arachidonic acid or ADP. These results were confirmed using Panx1-/- platelets. Natural variations have been described in the human Panx1 gene, which are predicted to induce non-conservative amino acid substitutions in its coding sequence. Healthy subjects homozygous for Panx1-400C, display enhanced platelet reactivity in response to collagen compared with those bearing the Panx1-400A allele. Conversely, the frequency of Panx1-400C homozygotes was increased among cardiovascular patients with hyper-reactive platelets compared with patients with hypo-reactive platelets. Exogenous expression of polymorphic Panx1 channels in a Panx-deficient cell line revealed increased basal and stimulated ATP release from cells transfected with Panx1-400C channels compared with Panx1-400A expressing transfectants. In conclusion, we demonstrate a specific role for Panx1 channels in the signalling pathway leading to collagen-induced platelet aggregation. Our study further identifies for the first time an association between a Panx1-400A>C genetic polymorphism and collagen-induced platelet reactivity. The Panx1-400C variant encodes for a gain-of-function channel that may adversely affect atherothrombosis by specifically enhancing collagen-induced ATP release and platelet aggregation.
Asunto(s)
Colágeno/farmacología , Conexinas/genética , Proteínas del Tejido Nervioso/genética , Agregación Plaquetaria/fisiología , Polimorfismo de Nucleótido Simple , Adenosina Difosfato/farmacología , Adenosina Trifosfato/metabolismo , Adolescente , Adulto , Alelos , Sustitución de Aminoácidos , Animales , Ácido Araquidónico/farmacología , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/genética , Conexinas/deficiencia , Conexinas/fisiología , Frecuencia de los Genes , Genotipo , Humanos , Masculino , Mefloquina/farmacología , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/fisiología , Fragmentos de Péptidos/farmacología , Agregación Plaquetaria/efectos de los fármacos , Probenecid/farmacología , Antagonistas del Receptor Purinérgico P2X/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Transfección , Adulto JovenRESUMEN
Junctional adhesion molecules (JAMs) are a family of adhesion molecules localized at the tight junction of polarized cells and on the cell surface of leukocytes. The last 20 years of research in this field has shown that several members of the family play an important role in the regulation of cell polarity, endothelium permeability and leukocytes migration. They mediate these pleiotropic functions through a multitude of homophilic and heterophilic interactions with intrafamily and extrafamily partners. In this article, we review the current status of the JAM family and highlight their functional role in tight junction dynamics and leukocyte transmigration.
Asunto(s)
Moléculas de Adhesión de Unión/fisiología , Uniones Estrechas/fisiología , Animales , Movimiento Celular/fisiología , Humanos , Leucocitos/fisiologíaRESUMEN
Atherosclerosis is a complex chronic inflammatory and metabolic disease that involves the collaboration of several cellular components of the immune system and results in thickening of the arterial wall. Atherosclerosis is also the primary cause of coronary artery and cerebrovascular diseases. A multitude of immune cell subsets, soluble molecules such as chemokines and cytokines, and circulating lipids play pivotal roles in atherosclerosis development. In this review, we highlight the role of the immune system in the course of atherosclerotic disease development and discuss the mechanisms involved.
Asunto(s)
Aterosclerosis/inmunología , Animales , Aterosclerosis/tratamiento farmacológico , Humanos , Factores Inmunológicos/farmacología , Factores Inmunológicos/uso terapéutico , Leucocitos/efectos de los fármacos , Leucocitos/inmunología , Leucocitos/metabolismoRESUMEN
The infiltration of inflammatory cells into the pancreas is an early and central event in acute pancreatitis that promotes local injury and systemic complications of the disease. Recent research has yielded the important finding that resident cells of the pancreas (particularly acinar and pancreatic stellate cells) play a dynamic role in leukocyte attraction via secretion of chemokines and cytokines and expression of adhesion molecules. Significant progress has been made in recent years in our understanding of the role of leukocyte movement (adhesion to the blood vessel wall, transmigration through the blood vessel wall and infiltration into the parenchyma) in the pathophysiology of acute pancreatitis. This review discusses recent studies and describes the current state of knowledge in the field. It is clear that detailed elucidation of the numerous processes in the inflammatory cascade is an essential step towards the development of improved therapeutic strategies in acute pancreatitis. Studies to date suggest that combination therapy targeting different steps of the inflammatory cascade may be the treatment of choice for this disease.
Asunto(s)
Páncreas Exocrino/fisiología , Pancreatitis/inmunología , Enfermedad Aguda , Animales , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/inmunología , Movimiento Celular/fisiología , Quimiocinas/inmunología , Quimiotaxis de Leucocito , Matriz Extracelular/fisiología , Humanos , Leucocitos/inmunología , Páncreas Exocrino/inmunología , Páncreas Exocrino/patología , Pancreatitis/patologíaRESUMEN
Jam-C(-/-) mice exhibit growth retardation and multilobular pneumonia concomitant with poor survival of the mice under conventional housing conditions. The deficient mice present a mega-oesophagus and have altered airway responsiveness. In addition, the number of circulating granulocytes is increased in Jam-C(-/-) mice as compared to control animals. These phenotypes probably reflect the different functions of JAM-C expressed by endothelial and mesenchymal cells. Indeed, the deregulation in the number of circulating granulocytes is caused by the lack of JAM-C expression on endothelial cells since rescuing endothelial expression of the protein in the Jam-C(-/-) mice is sufficient to restore homeostasis. More importantly, the rescue of vascular JAM-C expression is accompanied by better survival of deficient mice, suggesting that endothelial expression of JAM-C is mandatory for animal survival from opportunistic infections and fatal pneumonia.
Asunto(s)
Moléculas de Adhesión Celular/deficiencia , Granulocitos/inmunología , Homeostasis/inmunología , Inmunoglobulinas/deficiencia , Pulmón/inmunología , Proteínas de la Membrana/deficiencia , Animales , Bronquios/inmunología , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/inmunología , Susceptibilidad a Enfermedades/inmunología , Células Endoteliales/inmunología , Acalasia del Esófago/inmunología , Acalasia del Esófago/fisiopatología , Esófago/inmunología , Esófago/fisiopatología , Inmunoglobulinas/análisis , Inmunoglobulinas/inmunología , Inmunohistoquímica/métodos , Recuento de Leucocitos , Pulmón/fisiopatología , Proteínas de la Membrana/análisis , Proteínas de la Membrana/inmunología , Ratones , Ratones Mutantes , Músculo Liso/inmunología , Neutrófilos/inmunología , Peritonitis/inmunología , Peritonitis/fisiopatología , Neumonía/inmunología , Receptores CXCR4/análisisRESUMEN
Differentiation of naïve B cells into plasma cells or memory cells occurs in the germinal centers (GCs) of lymph follicles or alternatively via a GC- and T-cell-independent pathway. It is currently assumed that B-cell lymphomas correlate to normal B-cell differentiation stages, but the precise correlation of several B-cell lymphomas to these two pathways remains controversial. In the present report, we describe the junctional adhesion molecule C (JAM-C), currently identified at the cell-cell border of endothelial cells, as a new B-cell marker with a tightly regulated expression during B-cell differentiation. Expression of JAM-C in tonsils allows distinction between two CD27+ B-cell subpopulations: JAM-C- GC B cells and JAM-C+ non-germinal B cells. The expression of JAM-C in different B-cell lymphomas reveals a disease-specific pattern and allows a clear distinction between JAM-C- lymphoproliferative syndromes (chronic lymphocytic leukemia, mantle cell lymphoma and follicular lymphoma) and JAM-C+ ones (hairy cell leukemia, marginal zone B-cell lymphoma). Therefore, we propose JAM-C as a new identification tool in B-cell lymphoma diagnosis.
Asunto(s)
Linfocitos B/citología , Moléculas de Adhesión Celular/análisis , Centro Germinal/citología , Leucemia de Células B/diagnóstico , Linfoma de Células B/diagnóstico , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Biomarcadores de Tumor , Humanos , Leucemia de Células B/patología , Linfoma de Células B/patologíaRESUMEN
The recruitment of inflammatory cells contributes significantly to tissue injury in acute pancreatitis. This process implies several molecular interactions between circulating and endothelial cells. The adhesion molecule junctional adhesion molecule C (JAM-C) is involved in leukocyte transendothelial migration and it can form homophilic (JAM-C/JAM-C) and heterophilic interactions with the leukocyte integrin alpha(M)beta(2). In this study, the effect of early administration of monoclonal antibodies directed against JAM-C in cerulein-induced acute pancreatitis was assessed. This reagent significantly blocked influx of leukocytes, release of serum amylase, secretion of inflammatory cytokines, and acinar cell necrosis. These effects were rapid and protected against tissue injury throughout the duration of the model. Conversely, cerulein-induced acute pancreatitis was more severe in transgenic mice overexpressing JAM-C on endothelial cells under the control of the Tie2 promoter. It is proposed that JAM-C expressed by endothelial cells contributes to the pathophysiology of acute pancreatitis and could be considered a target for clinical applications.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Células Endoteliales/metabolismo , Inmunoglobulinas/fisiología , Proteínas de la Membrana/fisiología , Pancreatitis/metabolismo , Enfermedad Aguda , Amilasas/sangre , Animales , Anticuerpos Monoclonales/uso terapéutico , Western Blotting/métodos , Moléculas de Adhesión Celular/inmunología , Ceruletida , Quimiotaxis de Leucocito , Edema , Células Endoteliales/patología , Inmunoglobulinas/inmunología , Inmunohistoquímica/métodos , Interleucina-6/sangre , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Animales , Necrosis , Páncreas/inmunología , Páncreas/patología , Pancreatitis/sangre , Pancreatitis/patologíaRESUMEN
Endothelial cells express a diverse and exquisite array of adhesion molecules and cell surface receptors. Adhesion molecules expressed on endothelial cells not only maintain structural integrity of the vasculature, but also mediate more dynamic processes such as the highly regulated movement of leukocytes from free flow into different tissue compartments. Recent studies have focused on the molecular processes that mediate endothelial cell function and their ability to respond rapidly to changes in their immediate microenvironment, as well as maintaining routine cell trafficking through specialist tissue compartments. Adhesion molecules expressed on the endothelium mediate the movement of leukocytes into the underlying extravasculature to mediate a diverse array of functions including immune effector responses, cellular interactions in specialist lymphatic microenvironments and recirculation back into the vasculature. The true diversity and capacity of adhesion molecules capable of being expressed on the endothelium is now beginning to emerge, demonstrating new levels of complexity as specialist subsets of endothelium are characterised that define specific, yet diverse functions. In this chapter, the role of cell adhesion molecules in mediating endothelial cell function is discussed, from how their different physiochemical properties contribute to function, to how specific ligand interactions expressed on leukocyte cell populations contribute to functions ranging from constitutive cell trafficking to inflammation.
RESUMEN
Endothelial cells are linked to each other through intercellular junctional complexes that regulate the barrier and fence function of the vascular wall. The nature of these intercellular contacts varies with the need for permeability: For example, in brain the impervious blood-brain barrier is maintained by "tight" contacts between endothelial cells. By contrast, in high endothelial venules (HEVs), where lymphocytes continuously exit the bloodstream, the contacts are generally leaky. The precise molecular components that define the type of junction remain to be characterized. An immunoglobulin superfamily molecule named JAM-2, specifically expressed in lymphatic endothelial cells and HEVs, was recently identified. JAM-3 was cloned and characterized in the current study, and JAM-1, -2, and -3 were shown to form a novel protein family belonging to the larger cortical thymocyte Xenopus (CTX) molecular family. Using antibodies specific for each of the 3 family members, their specific participation in different types of cell-cell contact in vivo and their specific and differential localization in lateral contacts or tight junctions were demonstrated. Furthermore, it was shown that JAM-1 and JAM-2 differentially regulate paracellular permeability, suggesting that the presence of JAM-1, -2, or -3 in vascular junctions may play a role in regulating vascular function in vivo.
Asunto(s)
Moléculas de Adhesión Celular , Endotelio Vascular/ultraestructura , Expresión Génica , Inmunoglobulinas/genética , Uniones Intercelulares/fisiología , Proteínas de la Membrana/genética , Receptores de Superficie Celular , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Northern Blotting , Química Encefálica , Línea Celular , Permeabilidad de la Membrana Celular , Embrión de Mamíferos , Endotelio Vascular/química , Proteínas Fluorescentes Verdes , Inmunoglobulinas/análisis , Inmunoglobulinas/fisiología , Inmunohistoquímica , Riñón/química , Proteínas Luminiscentes/genética , Proteínas de la Membrana/análisis , Proteínas de la Membrana/fisiología , Ratones , Datos de Secuencia Molecular , Filogenia , ARN Mensajero/análisis , Proteínas Recombinantes de Fusión , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN , Distribución Tisular , TransfecciónRESUMEN
Integrin alpha(v)beta(3) is expressed by newly formed blood vessels in diseased and neoplastic tissue and can therefore be used as a marker for angiogenesis. We investigated its expression on the vasculature of 40 colon carcinomas using the anti-alpha(v)beta(3)-specific monoclonal antibody LM609. The average relapse-free interval and overall survival in patients suffering from colon carcinomas with high vascular expression of alpha(v)beta(3) integrin was significantly reduced compared with that in patients with low alpha(v)beta(3) integrin expressing tumor vasculature. Moreover, the expression level of alpha(v)beta(3) integrin correlated with the presence of liver metastases. In conclusion, we propose vascular expression of alpha(v)beta(3) integrin as a prognostic indicator for colon carcinoma.
Asunto(s)
Neoplasias del Colon/patología , Receptores de Vitronectina/biosíntesis , Anciano , Neoplasias del Colon/metabolismo , Endotelio Vascular/química , Endotelio Vascular/patología , Femenino , Humanos , Inmunohistoquímica , Neoplasias Hepáticas/química , Neoplasias Hepáticas/secundario , Masculino , Melanoma/metabolismo , Melanoma/secundario , Persona de Mediana Edad , Placenta/química , Placenta/patología , Análisis de Supervivencia , Molécula 1 de Adhesión Celular Vascular/análisisRESUMEN
HEMCAM/gicerin, an immunoglobulin superfamily protein, is involved in homophilic and heterophilic adhesion. It interacts with NOF (neurite outgrowth factor), a molecule of the laminin family. Alternative splicing leads to mRNAs coding for HEMCAM with a short (HEMCAM-s) or a long cytoplasmic tail (HEMCAM-l). To investigate the cellular function of these two variants, we stably transfected murine fibroblasts with either form of HEMCAM. Expression of each isoform of this protein in L cells delayed proliferation and modified their adhesion properties to purified extracellular matrix proteins. Expression of either HEMCAM-s or HEMCAM-l inhibited integrin-dependent adhesion and spreading of fibroblasts to laminin 1, showing that this phenomenon did not depend on the cytoplasmic region. By contrast, L-cell adhesion and spreading to fibronectin depended on the HEMCAM isoform expressed. Flow cytometry and immunoprecipitation studies revealed that the expression of HEMCAM downregulated expression of the laminin-binding integrins alpha3beta1, alpha6beta1 and alpha7beta1, and fibronectin receptor alpha5beta1 from the cell surface. Semi-quantitative PCR and northern blot experiments showed that the expression of alpha6beta1 integrin modified by HEMCAM occurred at a translation or maturation level. Thus, our data demonstrate that HEMCAM regulates fibroblast adhesion by controlling beta1 integrin expression.
Asunto(s)
Antígenos CD , Proteínas Aviares , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Glicoproteínas de Membrana , Moléculas de Adhesión de Célula Nerviosa , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Antígeno CD146 , Adhesión Celular/fisiología , División Celular/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Embrión de Pollo , Regulación hacia Abajo/fisiología , Fibroblastos/citología , Fibroblastos/metabolismo , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica , Humanos , Integrina alfa6beta1 , Integrinas/genética , Integrinas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , ARN Mensajero/análisis , Homología de Secuencia de Aminoácido , Transcripción Genética/fisiología , TransfecciónRESUMEN
Integrins are implicated in cell adhesion, migration and homeostasis. An important feature is their ability to adopt different affinity states that can be regulated by a variety of intra- and extracellular factors. To study affinity modulation of the integrin ectodomain by extracellular factors, we produced a soluble recombinant form of mouse integrin alphavbeta3 in a mammalian expression system and isolated it to purity. We show that the two transmembrane truncated integrin subunits stably associate to form a functional receptor, soluble recombinant alphavbeta3. The affinity of this receptor for its ligands vitronectin, fibronectin and fibrinogen can be modulated by the divalent cations magnesium, calcium and manganese. Most importantly, we found that a cyclic RGD-peptide has a biphasic effect on rsalphavbeta3and native purified alphavbeta3, with an antagonistic phase at high concentrations, and an agonistic phase at low concentrations. This integrin superactivation by low antagonist concentrations is shown in binding of sralphavbeta3 to immobilized ligands by ELISA, and in adhesion of cells that express the chimaeric integrin ligand KISS31 to immobilized rsalphavbeta3 and native purified alphavbeta3. Our results indicate that low concentrations of the ligand mimetic cyclo-RGD can result in superactivation of the extracellular domain of integrin alphavbeta3 to a comparable level as activation by manganese.
Asunto(s)
Adhesión Celular/efectos de los fármacos , Oligopéptidos/administración & dosificación , Receptores de Vitronectina/agonistas , Receptores de Vitronectina/antagonistas & inhibidores , Proteínas Recombinantes/biosíntesis , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Modelos Biológicos , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/genéticaRESUMEN
The immune system consists of a complex collection of leukocytes and dendritic cells that surveys most tissues in the body for the appearance of foreign antigens. For an efficient immune response, the interaction and co-localization of antigen-presenting cells, costimulatory helper cells and effector cells are crucial parameters. Therefore, the migration routes of antigen-presenting cells and potential antigen-specific lymphocytes merge in secondary lymphoid organs in order to increase the likelihood and speed of a lymphocyte finding its cognate antigen. Additionally, antigen-primed effector cells are directed to the tissue where they are most likely to encounter their cognate antigen. This highly organized and efficient antigen encounter is based on a continuous recirculation of antigen-specific lymphocytes between blood, peripheral tissue, and secondary lymphoid organs. Moreover, the efficacy of the immune system is further increased by the ability of different lymphocyte subsets to recirculate only through distinct tissues. The scope of this review is to outline the concept and mechanisms of lymphocyte homing and recirculation and to discuss the significance for the immune defense. Current models in leukocyte homing and recirculation and the underlying molecular functions of implicated cell adhesion molecules, chemokines, and chemokine receptors are discussed.
Asunto(s)
Linfocitos/inmunología , Receptores Mensajeros de Linfocitos/fisiología , Animales , Antígenos de Diferenciación Mielomonocítica/inmunología , Moléculas de Adhesión Celular/fisiología , Movimiento Celular/inmunología , Citocinas/inmunología , Endotelio Vascular/inmunología , Humanos , Tejido Linfoide/inmunología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/inmunologíaRESUMEN
Stem cell factor (also known as mast cell growth factor and kit-ligand) is a transmembrane growth factor with a highly conserved cytoplasmic domain. Basolateral membrane expression in epithelia and persistent cell surface exposure of stem cell factor are required for complete biological activity in pigmentation, fertility, learning, and hematopoiesis. Here we show by site-directed mutagenesis that the cytoplasmic domain of stem cell factor contains a monomeric leucine-dependent basolateral targeting signal. N-terminal to this motif, a cluster of acidic amino acids serves to increase the efficiency of basolateral sorting mediated by the leucine residue. Hence, basolateral targeting of stem cell factor requires a mono-leucine determinant assisted by a cluster of acidic amino acids. This mono-leucine determinant is functionally conserved in colony-stimulating factor-1, a transmembrane growth factor related to stem cell factor. Furthermore, this leucine motif is not capable of inducing endocytosis, allowing for persistent cell surface expression of stem cell factor. In contrast, the mutated cytoplasmic tail found in the stem cell factor mutant Mgf(Sl17H) induces constitutive endocytosis by a motif that is related to signals for endocytosis and lysosomal targeting. Our findings therefore present mono-leucines as a novel type of protein sorting motif for transmembrane growth factors.
Asunto(s)
Membrana Celular/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factor de Células Madre/genética , Factor de Células Madre/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Pollos , Secuencia de Consenso , Perros , Endocitosis , Proteínas Fluorescentes Verdes , Leucina , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Lisosomas/efectos de los fármacos , Lisosomas/fisiología , Ratones , Datos de Secuencia Molecular , Mutación , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Factor de Células Madre/química , Transfección , UrodelosRESUMEN
Cell-cell contacts are essential for morphogenesis and tissue function and play a vital role in mediating endothelial cohesion within the vascular system during vessel growth and organization. We identified a novel junctional adhesion molecule, named JAM-2, by a selective RNA display method, which allowed identification of transcripts encoding immunoglobulin superfamily molecules regulated during coculture of endothelial cells with tumor cells. The JAM-2 transcript is highly expressed during embryogenesis and is detected in lymph node and Peyer's patches RNA of adult mice. Accordingly, antibodies specific for JAM-2 stain high endothelial venules and lymphatic vessels in lymphoid organs, and vascular structures in the kidney. Using real time video microscopy, we show that JAM-2 is localized within minutes to the newly formed cell-cell contact. The role of the protein in the sealing of cell-cell contact is further suggested by the reduced paracellular permeability of cell monolayer transfected with JAM-2 cDNA, and by the localization of JAM-2 to tight junctional complexes of polarized cells. Taken together, our results suggest that JAM-2 is a novel vascular molecule, which participates in interendothelial junctional complexes.
Asunto(s)
Moléculas de Adhesión Celular/aislamiento & purificación , Endotelio Linfático/química , Endotelio Vascular/química , Inmunoglobulinas/aislamiento & purificación , Proteínas de la Membrana/aislamiento & purificación , Receptores de Superficie Celular , Uniones Estrechas/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Moléculas de Adhesión Celular/genética , Compartimento Celular , Polaridad Celular , Endotelio Linfático/ultraestructura , Endotelio Vascular/ultraestructura , Inmunoglobulinas/genética , Riñón/irrigación sanguínea , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Uniones Estrechas/ultraestructura , Distribución TisularRESUMEN
Integrins are cell-substrate adhesion molecules that provide the essential link between the actin cytoskeleton and the extracellular matrix during cell migration. We have analyzed alphaVbeta3-integrin dynamics in migrating cells using a green fluorescent protein-tagged beta3-integrin chain. At the cell front, adhesion sites containing alphaVbeta3-integrin remain stationary, whereas at the rear of the cell they slide inward. The integrin fluorescence intensity within these different focal adhesions, and hence the relative integrin density, is directly related to their mobility. Integrin density is as much as threefold higher in sliding compared with stationary focal adhesions. High intracellular tension under the control of RhoA induced the formation of high-density contacts. Low-density adhesion sites were induced by Rac1 and low intracellular tension. Photobleaching experiments demonstrated a slow turnover of beta3-integrins in low-density contacts, which may account for their stationary nature. In contrast, the fast beta3-integrin turnover observed in high-density contacts suggests that their apparent sliding may be caused by a polarized renewal of focal contacts. Therefore, differential acto-myosin-dependent integrin turnover and focal adhesion densities may explain the mechanical and behavioral differences between cell adhesion sites formed at the front, and those that move in the retracting rear of migrating cells.
Asunto(s)
Adhesiones Focales/fisiología , Receptores de Vitronectina/metabolismo , Células Cultivadas , Citometría de Flujo , Homeostasis , Humanos , Microscopía Fluorescente , Microscopía por Video , Receptores de Vitronectina/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Intraepithelial lymphocytes (IEL) of the small intestine are anatomically positioned to be in the first line of cellular defense against enteric pathogens. Therefore, determining the origin of these cells has important implications for the mechanisms of T cell maturation and repertoire selection. Recent evidence suggests that murine CD8 alpha alpha intestinal IELs (iIELs) can mature and undergo selection in the absence of a thymus. We analyzed IEL origin by cell transfer, using two congenic chicken strains. Embryonic day 14 and adult thymocytes did not contain any detectable CD8 alpha alpha T cells. However, when TCR(+) thymocytes were injected into congenic animals, they migrated to the gut and developed into CD8alphaalpha iIELs, while TCR(-) T cell progenitors did not. The TCR V beta 1 repertoire of CD8 alpha alpha(+) TCR V beta 1(+) iIELs contained only part of the TCR V beta 1 repertoire of total iIELs, and it exhibited no new members compared with CD8(+) T cells in the thymus. This indicated that these T cells emigrated from the thymus at an early stage in their developmental process. In conclusion, we show that while CD8 alpha alpha iIELs originate in the thymus, T cells acquire the expression of CD8 alpha alpha homodimers in the gut microenvironment.
Asunto(s)
Antígenos CD8/biosíntesis , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Timo/citología , Animales , Secuencia de Bases , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Movimiento Celular/genética , Movimiento Celular/inmunología , Senescencia Celular/genética , Senescencia Celular/inmunología , Embrión de Pollo , Pollos , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Mucosa Intestinal/citología , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/biosíntesis , Subgrupos de Linfocitos T/citología , Timo/inmunología , Timo/metabolismoAsunto(s)
Proteínas Aviares , Proteínas Fúngicas , Células Madre Hematopoyéticas/fisiología , Linfocitos T/fisiología , Animales , Antígenos de Superficie/fisiología , Antígeno CD146 , Moléculas de Adhesión Celular/fisiología , Diferenciación Celular , Movimiento Celular , Embrión de Pollo , Quitinasas/fisiología , Proteínas de Unión al ADN/fisiología , Antígenos de Histocompatibilidad Clase II/fisiología , Receptores de Hialuranos/fisiología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/fisiología , Proteínas Proto-Oncogénicas c-kit/fisiología , Factores de Transcripción/fisiologíaRESUMEN
T-cell progenitors in the embryonic bone marrow express the tyrosine kinase receptor c-kit. RR5, an anti-MHC class II beta chain monoclonal antibody, subdivides this c-kit positive population. Intrathymic transfer experiments showed that most of the T-cell progenitors belong to the MHC class II(+)/c-kit(+) bone marrow population in the embryo and young adult. On transplantation, these bone marrow progenitors lose this expression and differentiate into CD4 CD8 T lymphocytes. In contrast, erythroid progenitors are restricted to the MHC class II(-)/c-kit(+) population. The MHC class II(+)/c-kit(+) pro-T cells are metabolically active, because they stain brightly with rhodamin 123. Their cyclin A and B expression level suggests that they are in the mitotic phase of the cell cycle. Thus, we define an easy sorting protocol, which allows enrichment of T-cell progenitors from total bone marrow hemopoietic cells. (Blood. 2000;96:3988-3990)