RESUMEN
BACKGROUND: Successful immunotherapy will require alteration of the tumour microenvironment and/or decreased immune suppression. Tumour-associated macrophages (TAMs) are one major factor affecting tumour microenvironment. We hypothesised that altering TAM phenotype would augment the efficacy of immunotherapy. METHODS: We and others have reported that 5,6-Dimethylxanthenone-4-acetic-acid (DMXAA, Vadimezan) has the ability to change TAM phenotypes, inducing a tumour microenvironment conducive to antitumour immune responses. We therefore combined DMXAA with active immunotherapies, and evaluated anti-tumour efficacy, immune cell phenotypes (flow cytometry), and tumour microenvironment (RT-PCR). RESULTS: In several different murine models of immunotherapy for lung cancer, DMXAA-induced macrophage activation significantly augmented the therapeutic effects of immunotherapy. By increasing influx of neutrophils and anti-tumour (M1) macrophages to the tumour, DMXAA altered myeloid cell phenotypes, thus changing the intratumoural M2/non-M2 TAM immunoinhibitory ratio. It also altered the tumour microenvironment to be more pro-inflammatory. Modulating macrophages during immunotherapy resulted in increased numbers, activity, and antigen-specificity of intratumoural CD8(+) T cells. Macrophage depletion reduced the effect of combining immunotherapy with macrophage activation, supporting the importance of TAMs in the combined effect. CONCLUSION: Modulating intratumoural macrophages dramatically augmented the effect of immunotherapy. Our observations suggest that addition of agents that activate TAMs to immunotherapy should be considered in future trials.
Asunto(s)
Adenocarcinoma Bronquioloalveolar/terapia , Carcinoma Pulmonar de Lewis/terapia , Inmunoterapia , Neoplasias Pulmonares/terapia , Activación de Macrófagos/efectos de los fármacos , Microambiente Tumoral/inmunología , Xantonas/uso terapéutico , Adenocarcinoma Bronquioloalveolar/inmunología , Adenocarcinoma Bronquioloalveolar/patología , Animales , Antineoplásicos/uso terapéutico , Linfocitos T CD8-positivos/inmunología , Carcinoma Pulmonar de Lewis/inmunología , Carcinoma Pulmonar de Lewis/patología , Terapia Combinada , Femenino , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células Mieloides/citología , Células Mieloides/inmunología , Neutrófilos/citología , Neutrófilos/inmunologíaRESUMEN
BACKGROUND: Species selectivity of DMXAA (5,6-dimethylxanthenone-4-acetic acid, Vadimezan) for murine cells over human cells could explain in part the recent disappointing phase III trials clinical results when preclinical studies were so promising. To identify analogues with greater human clinical potential, we compared the activity of xanthenone-4-acetic acid (XAA) analogues in murine or human cellular models. METHODS: Analogues with a methyl group systematically substituted at different positions of the XAA backbone were evaluated for cytokine induction in cultured murine or human leukocytes; and for anti-vascular effects on endothelial cells on matrigel. In vivo antitumour activity and cytokine production by stromal or cancer cells was measured in human A375 and HCT116 xenografts. RESULTS: Mono-methyl XAA analogues with substitutions at the seventh and eighth positions were the most active in stimulating human leukocytes to produce IL-6 and IL-8; and for inhibition of tube formation by ECV304 human endothelial-like cells, while 5- and 6-substituted analogues were the most active in murine cell systems. CONCLUSION: Xanthenone-4-acetic acid analogues exhibit extreme species selectivity. Analogues that are the most active in human systems are inactive in murine models, highlighting the need for the use of appropriate in vivo animal models in selecting clinical candidates for this class of compounds.
Asunto(s)
Antineoplásicos/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Xantenos/farmacología , Xantonas/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colágeno/metabolismo , Combinación de Medicamentos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Laminina/metabolismo , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Ratones , Ratones Desnudos , Proteoglicanos/metabolismo , Xantenos/químicaRESUMEN
The molecular nature of cell adhesion mediated by platelet/endothelial cell adhesion molecule 1 (PECAM-1; CD31) was examined using stably transfected L cells in a PECAM-dependent aggregation assay. This adhesion was temperature sensitive and divalent cation dependent, with Mg2+ supporting aggregation to a greater degree than Ca2+. PECAM-dependent aggregation was heterophilic: PECAM-1 transfectants bound as readily to control-transfected L cells as to other PECAM-1 transfectants, demonstrating that a molecule endogenously expressed on the L cells serves as the ligand for PECAM in this system and presumably substitutes for the natural human ligand.
Asunto(s)
Antígenos de Diferenciación Mielomonocítica/fisiología , Adhesión Celular/fisiología , Animales , Cationes , Cinética , Células L , Ratones , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , Temperatura , TransfecciónRESUMEN
Asthma is a disease of airway inflammation and hyperreactivity that is associated with a lymphocytic infiltrate in the bronchial submucosa. The interactions between infiltrating T lymphocytes with cellular and extracellular matrix components of the airway and the consequences of these interactions have not been defined. We demonstrate the constitutive expression of CD44 on human airway smooth muscle (ASM) cells in culture as well as in human bronchial tissue transplanted into severe combined immunodeficient mice. In contrast, basal levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression are minimal but are induced on ASM by inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha). Activated, but not resting T cells, adhere to cultured ASM; stimulation of the ASM with TNF-alpha enhanced this adhesion. Adhesion was partially blocked by monoclonal antibodies (mAb) specific for lymphocyte function-associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4) on T cells and ICAM-1 and VCAM-1 on ASM cells. The observed integrin-independent adhesion was mediated by CD44/hyaluronate interactions as it was inhibited by anti-CD44 mAb 5F12 and by hyaluronidase. Furthermore, the adhesion of activated T lymphocytes induced DNA synthesis in growth-arrested ASM cells. Thus, the interaction between T cells and ASM may provide insight into the mechanisms that induce bronchial inflammation and possibly ASM cell hyperplasia seen in asthma.
Asunto(s)
Bronquios/citología , Proteínas Portadoras/fisiología , ADN/biosíntesis , Integrinas/fisiología , Músculo Liso/metabolismo , Receptores de Superficie Celular/fisiología , Receptores Mensajeros de Linfocitos/fisiología , Linfocitos T/fisiología , Tráquea/citología , Animales , Bronquios/metabolismo , Adhesión Celular , Moléculas de Adhesión Celular/análisis , Comunicación Celular , Células Cultivadas , Humanos , Receptores de Hialuranos , Molécula 1 de Adhesión Intercelular , Ratones , Ratones SCID , Tráquea/metabolismo , Molécula 1 de Adhesión Celular VascularRESUMEN
The CD31 (platelet endothelial cell adhesion molecule-1 [PECAM-1]/endothelial cell adhesion molecule [endoCAM]) molecule expressed on leukocytes, platelets, and endothelial cells is postulated to mediate adhesion to endothelial cells and thereby function in immunity, inflammation, and wound healing. We report the following novel features of CD31 which suggests a role for it in adhesion amplification of unique T cell subsets: (a) engagement of CD31 induces the adhesive function of beta 1 and beta 2 integrins; (b) adhesion induction by CD31 immunoglobulin G (IgG) monoclonal antibodies (mAbs) is sensitive, requiring only bivalent mAb; (c) CD31 mAb induces adhesion rapidly, but it is transient; (d) unique subsets of CD4+ and CD8+ T cells express CD31, including all naive (CD45RA+) CD8 T cells; and (e) CD31 induction is selective, inducing adhesive function of beta 1 integrins, particularly very late antigen-4, more efficiently than the beta 2 integrin lymphocyte function-associated antigen-1. Conversely, CD3 is more effective in inducing beta 2-mediated adhesion. Taken together, these findings indicate that unique T cell subsets express CD31, and CD31 has the capacity to induce integrin-mediated adhesion of T cells in a sensitive and selective fashion. We propose that, in collaboration with other receptors/ligands, CD31 functions in an "adhesion cascade" by amplifying integrin-mediated adhesion of CD31+ T cells to other cells, particularly endothelial cells.
Asunto(s)
Antígenos de Diferenciación Mielomonocítica/fisiología , Integrinas/fisiología , Subgrupos de Linfocitos T/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Antígenos de Diferenciación de Linfocitos T/fisiología , Complejo CD3 , Adhesión Celular , Moléculas de Adhesión Celular/fisiología , Antígenos de Histocompatibilidad/análisis , Humanos , Integrina beta1 , Antígenos Comunes de Leucocito , Antígeno-1 Asociado a Función de Linfocito/fisiología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , Receptores de Antígenos de Linfocitos T/fisiología , Receptores de Antígeno muy Tardío/fisiología , Linfocitos T/fisiología , Molécula 1 de Adhesión Celular VascularRESUMEN
Platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31) plays an active role in the process of leukocyte migration through cultured endothelial cells in vitro and anti-PECAM-1 antibodies (Abs) inhibit accumulation of leukocytes into sites of inflammation in vivo. Despite the latter, it is still not clear at which stage of leukocyte emigration in vivo PECAM-1 is involved. To address this point directly, we studied the effect of an anti-PECAM-1 Ab, recognizing rat PECAM-1, on leukocyte responses within rat mesenteric microvessels using intravital microscopy. In mesenteric preparations activated by interleukin (IL)-1 beta, the anti-PECAM-1 Ab had no significant effect on the rolling or adhesion of leukocytes, but inhibited their migration into the surrounding extravascular tissue in a dose-dependent manner. Although in some vessel segments these leukocytes had come to a halt within the vascular lumen, often the leukocytes appeared to be trapped within the vessel wall. Analysis of these sections by electron microscopy revealed that the leukocytes had passed through endothelial cell junctions but not the basement membrane. In contrast to the effect of the Ab in mesenteric preparations treated with IL-1 beta, leukocyte extravasation induced by topical or intraperitoneal administration of the chemotactic peptide formyl-methionyl-leucyl-phenylalanine was not inhibited by the anti-PECAM-1 Ab. These results directly demonstrate a role for PECAM-1 in leukocyte extravasation in vivo and indicate that this involvement is selective for leukocyte extravasation elicited by certain inflammatory mediators. Further, our findings provide the first in vivo indication that PECAM-1 may have an important role in triggering the passage of leukocytes through the perivascular basement membrane.
Asunto(s)
Antígenos de Diferenciación Mielomonocítica/fisiología , Membrana Basal/fisiología , Moléculas de Adhesión Celular/fisiología , Endotelio Vascular/fisiología , Inflamación/patología , Leucocitos/citología , Animales , Anticuerpos Monoclonales , Adhesión Celular , Interleucina-1/farmacología , Masculino , Mesenterio/citología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , Ratas , Ratas Sprague-Dawley , Grabación en VideoRESUMEN
Platelet/endothelial cell adhesion molecule (PECAM-1) is a cell adhesion molecule of the immunoglobulin superfamily that plays a role in a number of vascular processes including leukocyte transmigration through endothelium. The presence of a specific 19- amino acid exon within the cytoplasmic domain of PECAM-1 regulates the binding specificity of the molecule; specifically, isoforms containing exon 14 mediate heterophilic cell-cell aggregation while those variants missing exon 14 mediate homophilic cell-cell aggregation. To more precisely identify the region of exon 14 responsible for ligand specificity, a series of deletion mutants were created in which smaller regions of exon 14 were removed. After transfection into L cells, they were tested for their ability to mediate aggregation. For heterophilic aggregation to occur, a conserved 5-amino acid region (VYSEI in the murine sequence or VYSEV in the human sequence) in the mid-portion of the exon was required. A final construct, in which this tyrosine was mutated into a phenylalanine, aggregated in a homophilic manner when transfected into L cells. Inhibition of phosphatase activity by exposure of cells expressing wild type or mutant forms of PECAM-1 to sodium orthovanadate resulted in high levels of cytoplasmic tyrosine phosphorylation and led to a switch from heterophilic to homophilic aggregation. Our data thus indicate either loss of this tyrosine from exon 14 or its phosphorylation results in a change in ligand specificity from heterophilic to homophilic binding. Vascular cells could thus determine whether PECAM-1 functions as a heterophilic or homophilic adhesion molecule by processes such as alternative splicing or by regulation of the balance between tyrosine phosphorylation or dephosphorylation. Defining the conditions under which these changes occur will be important in understanding the biology of PECAM-1 in transmigration, angiogenesis, development, and other processes in which this molecule plays a role.
Asunto(s)
Plaquetas/química , Exones/fisiología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/química , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Empalme Alternativo/fisiología , Animales , Sitios de Unión/fisiología , Plaquetas/metabolismo , Citoplasma/química , Ligandos , Ratones , Mutagénesis/fisiología , Fosforilación , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Estructura Terciaria de Proteína , Sensibilidad y Especificidad , Tirosina/metabolismo , Vanadatos/farmacologíaRESUMEN
Cell-cell adhesion is controlled by many molecules found on the cell surface. In addition to the constituents of well-defined junctional structures, there are the molecules that are thought to play a role in the initial interactions of cells and that appear at precise times during development. These include the cadherins and cell adhesion molecules (CAMs). Representatives of these families of adhesion molecules have been isolated from most of the major tissues. The notable exception is the vascular endothelium. Here we report the identification of a cell surface molecule designated "endoCAM" (endothelial Cell Adhesion Molecule), which may function as an endothelial cell-cell adhesion molecule. EndoCAM is a 130-kD glycoprotein expressed on the surface of endothelial cells both in culture and in situ. It is localized to the borders of contiguous endothelial cells. It is also present on platelets and white blood cells. Antibodies against endoCAM prevent the initial formation of endothelial cell-cell contacts. Despite similarities in size and intercellular location, endoCAM does not appear to be a member of the cadherin family of adhesion receptors. The serologic and protease susceptibility characteristics of endoCAM are different from those of the known cadherins, including an endogenous endothelial cadherin. Although the precise biologic function of endoCAM has not been determined, it appears to be one of the molecules responsible for regulating endothelial cell-cell adhesion processes and may be involved in platelet and white blood cell interactions with the endothelium.
Asunto(s)
Antígenos de Diferenciación Mielomonocítica/aislamiento & purificación , Moléculas de Adhesión Celular/aislamiento & purificación , Adhesión Celular , Animales , Anticuerpos Monoclonales , Antígenos de Diferenciación Mielomonocítica/análisis , Plaquetas/análisis , Calcio/farmacología , Bovinos , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/farmacología , Células Cultivadas , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Técnica del Anticuerpo Fluorescente , Humanos , Sueros Inmunes , Uniones Intercelulares/análisis , Uniones Intercelulares/ultraestructura , Peso Molecular , Péptido Hidrolasas , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , Inhibidores de Proteasas , TripsinaRESUMEN
PECAM-1 is a 130-120-kD integral membrane glycoprotein found on the surface of platelets, at endothelial intercellular junctions in culture, and on cells of myeloid lineage. Previous studies have shown that it is a member of the immunoglobulin gene superfamily and that antibodies against the bovine form of this protein (endoCAM) can inhibit endothelial cell-cell interactions. These data suggest that PECAM-1 may function as a vascular cell adhesion molecule. The function of this molecule has been further evaluated by transfecting cells with a full-length PECAM-1 cDNA. Transfected COS-7, mouse 3T3 and L cells expressed a 130-120-kD glycoprotein on their cell surface that reacted with anti-PECAM-1 polyclonal and monoclonal antibodies. COS-7 and 3T3 cell transfectants formed cell-cell junctions that were highly enriched in PECAM-1, reminiscent of its distribution at endothelial cell-cell borders. In contrast, this protein remained diffusely distributed within the plasma membrane of PECAM-1 transfected cells that were in contact with mock transfectants. Mouse L cells stably transfected with PECAM-1 demonstrated calcium-dependent aggregation that was inhibited by anti-PECAM antibodies. These results demonstrate that PECAM-1 mediates cell-cell adhesion and support the idea that it may be involved in some of the interactive events taking place during thrombosis, wound healing, and angiogenesis.
Asunto(s)
Antígenos CD/fisiología , Antígenos de Diferenciación Mielomonocítica/fisiología , Plaquetas/citología , Moléculas de Adhesión Celular/fisiología , Endotelio Vascular/citología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Calcio/fisiología , Agregación Celular , Chlorocebus aethiops , Clonación Molecular , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Técnicas In Vitro , Uniones Intercelulares/ultraestructura , Ratones , Datos de Secuencia Molecular , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , ARN Mensajero/genética , TransfecciónRESUMEN
Integrin beta subunits combine with specific sets of alpha subunits to form functional adhesion receptors. The structure and binding properties of integrins suggest the presence of domains controlling at least three major functions: subunit association, ligand binding, and cytoskeletal interactions. To more carefully define structure/function relationships, a cDNA construct consisting of the extracellular domain of the avian beta 1 subunit and the cytoplasmic and transmembrane domains of the human beta 3 subunit was prepared and expressed in murine 3T3 cells. The resulting chimeric beta 1/3 subunit formed heterodimers with alpha subunits from the beta 1 subfamily, could not interact with alpha IIb from the beta 3 subfamily, was targeted to focal contacts, and formed functional complexes within the focal contacts. A second cDNA construct was prepared that coded for an avian beta 1 subunit without a transmembrane or cytoplasmic domain. This subunit was not found in association with an accompanying alpha subunit, nor was it found expressed on the cell surface. Instead, it accumulated in vesicles within the cytoplasm and was eventually shed from the cell. The results from studies of the behavior of these two cDNA constructs demonstrate that the transmembrane and cytoplasmic domains play no role in alpha subunit selection, that the cytoplasmic domain of beta 3 is capable of functioning in the context of alpha subunits with which it is not normally paired, and that both integrin subunits must be membrane associated for normal assembly and transport to cell surface adhesive structures.
Asunto(s)
Integrinas/química , Animales , Adhesión Celular , Citoplasma/ultraestructura , Proteínas del Citoesqueleto/metabolismo , Análisis Mutacional de ADN , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas Inmunológicas , Integrinas/fisiología , Integrinas/ultraestructura , Sustancias Macromoleculares , Estructura Molecular , Proteínas Recombinantes de Fusión , Relación Estructura-Actividad , Transfección , VinculinaRESUMEN
Although a biphasic dependence of cell migration speed on cell-substratum adhesiveness has been predicted theoretically, experimental data directly demonstrating a relationship between these two phenomena have been lacking. To determine whether an optimal strength of cell-substratum adhesive interactions exists for cell migration, we measured quantitatively both the initial attachment strength and migration speed of human smooth muscle cells (HSMCs) on a range of surface concentrations of fibronectin (Fn) and type IV collagen (CnIV). Initial attachment strength was measured in order to characterize short time-scale cell-substratum interactions, which may be representative of dynamic interactions involved in cell migration. The critical fluid shear stress for cell detachment, determined in a radial-flow detachment assay, increased linearly with the surface concentrations of adsorbed Fn and CnIV. The detachment stress required for cells on Fn, 3.6 +/- 0.2 x 10(-3) mu dynes/absorbed molecule, was much greater than that on CnIV, 5.0 +/- 1.4 x 10(-5) mu dynes/absorbed molecule. Time-lapse videomicroscopy of individual cell movement paths showed that the migration behavior of HSMCs on these substrates varied with the absorbed concentration of each matrix protein, exhibiting biphasic dependence. Cell speed reached a maximum at intermediate concentrations of both proteins, with optimal concentrations for migration at 1 x 10(3) molecules/micron2 and 1 x 10(4) molecules/micron2 on Fn and CnIV, respectively. These optimal protein concentrations represent optimal initial attachment strengths corresponding to detachment shear stresses of 3.8 mu dyne/micron2 on Fn and 1.5 mu dyne/micron2 on CnIV. Thus, while the optimal absorbed protein concentrations for migration on Fn and CnIV differed by an order of magnitude, the optimal initial attachment strengths for migration on these two proteins were very similar. Further, the same minimum strength of initial attachment, corresponding to a detachment shear stress of approximately 1 mu dyne/micron2, was required for movement on either protein. These results suggest that initial cell-substratum attachment strength is a central variable governing cell migration speed, able to correlate observations of motility on substrata differing in adhesiveness. They also demonstrate that migration speed depends in biphasic manner on attachment strength, with maximal migration at an intermediate level of cell-substratum adhesiveness.
Asunto(s)
Colágeno , Fibronectinas , Músculo Liso Vascular/citología , Adhesión Celular , Movimiento Celular , Células Cultivadas , HumanosRESUMEN
Bovine aortic endothelial cell (BAEC) attachments to laminin, fibronectin, and fibrinogen are inhibited by soluble arginine-glycine-aspartate (RGD)-containing peptides, and YGRGDSP activity is responsive to titration of either soluble peptide or matrix protein. To assess the presence of RGD-dependent receptors, immunoprecipitation and immunoblotting studies were conducted and demonstrated integrin beta 1, beta 3, and associated alpha subunits as well as a beta 1 precursor. Immunofluorescence of BAECs plated on laminin, fibronectin, and fibrinogen reveals different matrix-binding specificities of each of these integrin subclasses. By 1 h after plating, organization of beta 1 integrin into fibrillar streaks is influenced by laminin and fibronectin, whereas beta 3 integrin punctate organization is influenced by fibrinogen and the integrin spatial distribution changes with time in culture. In contrast, the nonintegrin laminin-binding protein LB69 only organizes after cell-substrate contact is well established several hours after plating. Migration of BAECs is also mediated by both integrin and nonintegrin matrix-binding proteins. Specifically, BAEC migration on laminin is remarkably sensitive to RGD peptide inhibition, and, in its presence, beta 1 integrin organization dissipates and reorganizes into perinuclear vesicles. However, RGD peptides do not alter LB69 linear organization during migration. Similarly, agents that block LB69--e.g., antibodies to LB69 as well as YIGSR-NH2 peptide--do not inhibit attachment of nonmotile BAECs to laminin. However, both anti-LB69 and YIGSR-NH2 inhibit late adhesive events such as spreading. Accordingly, we propose that integrin and nonintegrin extracellular matrix-binding protein organizations in BAECs are both temporally and spatially segregated during attachment processes. High affinity nonintegrin interaction with matrix may create necessary stable contacts for longterm attachment, while lower affinity integrins may be important for initial cell adhesion as well as for transient contacts of motile BAECs.
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Adhesión Celular , Endotelio Vascular/fisiología , Matriz Extracelular/fisiología , Integrinas/fisiología , Oligopéptidos/farmacología , Fosfatasa Ácida/metabolismo , Secuencia de Aminoácidos , Animales , Aorta , Bovinos , Adhesión Celular/efectos de los fármacos , Movimiento Celular , Células Cultivadas , Fibrinógeno/fisiología , Fibronectinas/fisiología , Técnica del Anticuerpo Fluorescente , Laminina/fisiología , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Oligopéptidos/metabolismoRESUMEN
Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a member of the immunoglobulin superfamily present on platelets, endothelial cells, and leukocytes that may function as a vascular cell adhesion molecule. The purpose of this study was to examine the role of the cytoplasmic domain in PECAM-1 function. To accomplish this, wild-type and mutated forms of PECAM-1 cDNA were transfected into murine fibroblasts and the functional characteristics of the cells analyzed. Wild-type PECAM-1 localized to the cell-cell borders of adjacently transfected cells and mediated heterophilic, calcium-dependent L-cell aggregation that was inhibitable by a polyclonal and two monoclonal anti-PECAM-1 antibodies. A mutant protein lacking the entire cytoplasmic domain did not support aggregation or move to cell-cell borders. In contrast, both forms of PECAM-1 with partially truncated cytoplasmic domains (missing either the COOH-terminal third or two thirds of the cytoplasmic domain) localized to cell-cell borders in 3T3 cells in a manner analogous to the distribution seen in cultured endothelial cells. L-cells expressing these mutants demonstrated homophilic, calcium-independent aggregation that was blocked by the polyclonal anti-PECAM-1 antibody, but not by the two bioactive monoclonal antibodies. Although changes in the cytoplasmic domain of other receptors have been shown to alter ligand-binding affinity, to our knowledge, PECAM-1 is the first example of a cell adhesion molecule where changes in the cytoplasmic domain result in a switch in the basic mechanism of adhesion leading to different ligand-binding specificity. Variations in the cytoplasmic domain could thus be a potential mechanism for regulating PECAM-1 activity in vivo.
Asunto(s)
Antígenos de Diferenciación Mielomonocítica/química , Moléculas de Adhesión Celular/química , Adhesión Celular , Glicoproteínas de Membrana/química , Células 3T3 , Animales , Antígenos de Diferenciación Mielomonocítica/metabolismo , Moléculas de Adhesión Celular/metabolismo , Compartimento Celular , Citoplasma/metabolismo , Ligandos , Glicoproteínas de Membrana/metabolismo , Ratones , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , Unión Proteica , Agregación de Receptores , Proteínas Recombinantes , Eliminación de Secuencia , Relación Estructura-Actividad , TransfecciónRESUMEN
During inflammation, neutrophils migrate from the vascular lumen into extravascular sites. In vitro assays have suggested that platelet-endothelial cell adhesion molecule-1 [PECAM-1 (CD31)], a member of the immunoglobulin superfamily, is required for the transmigration of neutrophils across endothelial monolayers. Antibody to human PECAM-1, which cross-reacts with rat PECAM-1, was found to block not only in vivo accumulation of rat neutrophils into the peritoneal cavity and the alveolar compartment of the lung but also neutrophil accumulation in human skin grafts transplanted onto immunodeficient mice. On the basis of these findings in three different models of inflammation, it appears that PECAM-1 is required for neutrophil transmigration in vivo and may thus be a potential therapeutic target.
Asunto(s)
Antígenos de Diferenciación Mielomonocítica/fisiología , Moléculas de Adhesión Celular/fisiología , Glicoproteínas de Membrana/fisiología , Neutrófilos/fisiología , Animales , Anticuerpos/farmacología , Antígenos de Diferenciación Mielomonocítica/inmunología , Moléculas de Adhesión Celular/inmunología , Movimiento Celular/fisiología , Quimiotaxis de Leucocito/fisiología , Endotelio/inmunología , Humanos , Enfermedades del Complejo Inmune/inmunología , Glicoproteínas de Membrana/inmunología , Ratones , Ratones SCID , Cavidad Peritoneal/citología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , Ratas , Trasplante de Piel/inmunología , Trasplante Heterólogo/inmunologíaRESUMEN
The eighth International Mesothelioma Interest Group (IMIG) meeting was held in Chicago, IL, United States, in 19-22 October 2006 to discuss mesothelioma - the cancer often linked to asbestos exposure. It is a very aggressive malignancy with a median survival of less than 1 year from diagnosis. Millions of people have been exposed worldwide to asbestos, especially during the second half of the twentieth century when asbestos use increased significantly. The tons of asbestos utilized in the past remain a health hazard for current and future generations because asbestos is difficult to be disposed off. This makes asbestos and mesothelioma research a public health issue in addition to a medical problem. Moreover, the very high costs of asbestos litigation have a significant impact on the whole economy. In the United States, up until 2001, defendant companies had paid 54 billion dollars in claims and estimated future liabilities ranged from 145 to 210 billion. Therefore, asbestos research is of great interest to a large audience that includes patients, millions of asbestos-exposed individuals, scientists, physicians, public health officials, politicians, unions of asbestos workers, lawyers and the public at large. During the past few years, there has been significant progress in understanding the process of mineral fiber carcinogenesis and mesothelioma pathogenesis. With improved understanding of the pathogenesis of mesothelioma, new diagnostic, preventive and therapeutic options are being developed. A total of 247 papers were presented at the IMIG: the abstracts of these presentations were published in Lung Cancer, Supplement 1, October 2006. Here, experts in different disciplines critically review some of the most exciting presentations of the IMIG meeting. The result is a comprehensive review of the research field of asbestos carcinogenesis and mesothelioma, and of the progress that has been made in recent years in both basic and clinical sciences.
Asunto(s)
Mesotelioma , Neoplasias Pleurales , Humanos , Mesotelioma/etiología , Mesotelioma/patología , Neoplasias Pleurales/etiología , Neoplasias Pleurales/patologíaRESUMEN
A series of immunological approaches was utilized to identify the molecules involved in cell-substratum adhesion of human endothelial cells (EC) derived from adult large vessels, fat capillaries, and umbilical veins. A polyclonal antibody prepared against partially purified extracellular matrix receptors disrupted adhesion of EC to a wide variety of substrates and identified four groups of glycoproteins migrating with apparent Mr of 150, 125, 110, and 95 kD in immunoprecipitation experiments. Specific monoclonal antibodies identified these proteins as members of the Integrin family of extracellular matrix receptors and included the alpha and beta chains of the fibronectin receptor (alpha 5/beta 1), a collagen receptor (alpha 2 beta 1), a multifunctional receptor that binds to fibronectin, collagen, and laminin (alpha 3/beta 1), as well as a receptor related to platelet IIb/IIIa (alpha v/beta 3). To directly test the importance of these molecules in cell-substratum adhesion, these proteins were purified by a combination of ion exchange, lectin affinity, and immunoaffinity chromatography and used to block the biological activity of the adhesion-disrupting polyclonal antibody. Immunofluorescence experiments further supported the role of these glycoproteins in adhesion. The GPIIb/IIIa-like receptor localized to well-formed adhesion plaques on EC plated on fibrinogen, but not on fibronectin, laminin, or type IV collagen. Receptors containing the beta 1 subunit were visualized as discontinuous fibrils which colocalized with fibronectin fibrils and actin stress fibers.
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Antígenos de Superficie/aislamiento & purificación , Adhesión Celular , Endotelio Vascular/metabolismo , Glicoproteínas de Membrana/aislamiento & purificación , Receptores Inmunológicos/aislamiento & purificación , Adulto , Animales , Anticuerpos Monoclonales/fisiología , Antígenos de Superficie/inmunología , Moléculas de Adhesión Celular , Cricetinae , Endotelio Vascular/fisiología , Matriz Extracelular/metabolismo , Humanos , Sueros Inmunes/farmacología , Integrinas , Glicoproteínas de Membrana/inmunología , Pruebas de Precipitina , RatasRESUMEN
The ability of circulating white blood cells to enter inflamed tissues is mediated by specific cell adhesion molecules thought to be expressed in a programmed and sequential manner to form an "adhesion cascade." Because of the complexity of this process, it is becoming increasingly important to develop in vivo models. Two major problems have limited the utility of current animal models. The first is the inability of many of the antibodies developed against cell adhesion molecules in human cell culture models to cross-react in animals. The second is the uncertainty in extrapolating animal (particularly rodent) findings to humans. To circumvent these problems, full thickness human skin grafts were transplanted onto immunodeficient (severe combined immunodeficient) mice. After 4-6 wk, the transplanted skin grafts closely resembled normal skin histologically and maintained their human vasculature as determined by immunohistochemical staining with human-specific endothelial cell markers. Intradermal injection of tumor necrosis factor-alpha resulted in the reversible upregulation of the leukocyte-endothelial adhesion molecules E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1, and in an active inflammatory reaction with migration of murine leukocytes into cytokine-injected areas. These results indicate that the severe combined immunodeficient mouse/human skin transplant model provides a useful in vivo system in which to study human endothelium during the process of inflammation.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Trasplante de Piel/fisiología , Adulto , Animales , Antígenos de Diferenciación Mielomonocítica/metabolismo , Moléculas de Adhesión Celular/análisis , Quimera , Selectina E , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Recién Nacido , Molécula 1 de Adhesión Intercelular , Leucocitos/citología , Leucocitos/efectos de los fármacos , Leucocitos/fisiología , Masculino , Ratones , Ratones SCID , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , Piel/irrigación sanguínea , Piel/efectos de los fármacos , Trasplante de Piel/inmunología , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular VascularRESUMEN
Integrins are a class of cell adhesion molecules that participate in cell-cell and cell-substratum interactions and are present on essentially all human cells. The distribution of nine different alpha and beta integrin subunits in human endometrial tissue at different stages of the menstrual cycle was determined using immunoperoxidase staining. Glandular epithelial cells expressed primarily alpha 2, alpha 3, and alpha 6 (collagen/laminin receptors), while stromal cells expressed predominantly alpha 5 (fibronectin receptor). The presence of alpha 1 on glandular epithelial cells was cycle specific, found only during the secretory phase. Expression of both subunits of the vitronectin receptor, alpha v beta 3, also underwent cycle specific changes on endometrial epithelial cells. Immunostaining for alpha v increased throughout the menstrual cycle, while the beta 3 subunit appeared abruptly on cycle day 20 on luminal as well as glandular epithelial cells. Discordant luteal phase biopsies (greater than or equal to 3 d "out of phase") from infertility patients exhibited delayed epithelial beta 3 immunostaining. These results demonstrate similarities, as well as specific differences, between endometrium and other epithelial tissues. Certain integrin moieties appear to be regulated within the cycling endometrium and disruption of integrin expression may be associated with decreased uterine receptivity and infertility.
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Endometrio/química , Integrinas/análisis , Ciclo Menstrual , Adulto , Femenino , Glicoproteínas/análisis , Humanos , Inmunohistoquímica , Infertilidad/metabolismo , VitronectinaRESUMEN
Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a member of the immunoglobulin superfamily that plays a role in a number of endothelial cell (EC) functions including migration, angiogenesis, and transmigration of leukocytes across endothelium. We postulated that one way PECAM-1 might exert its effects was by regulating intracellular EC levels of calcium. Using single-cell fluorometry, we found that engagement of PECAM-1 by mAbs induced a slow but sustained increase in intracellular calcium, both in EC and in an adherent PECAM-1-transfected cell line that models endothelium. Generation of this signal was specific for certain anti-PECAM-1 antibodies, required the presence of the cytoplasmic domain, depended on extracellular calcium and on tyrosine phosphorylation, but did not require cross-linking; in fact, calcium increases were stimulated by certain Fab fragments. Activation of EC by PECAM-1 also caused a time-dependent increase in prostacyclin release. Given the importance of intracellular calcium and prostacyclin release as signaling molecules, engagement of PECAM-1 during cell-cell interactions may alter a number of EC functions including secretion of vasoactive mediators.
Asunto(s)
Calcio/metabolismo , Endotelio Vascular/metabolismo , Epoprostenol/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Sitios de Unión , Cationes Bivalentes , Línea Celular , Endotelio Vascular/citología , Epítopos de Linfocito B/inmunología , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos Fab de Inmunoglobulinas/farmacología , Ratones , Fosforilación , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Relación Estructura-Actividad , Transfección , Tirosina/metabolismo , Venas Umbilicales/citologíaRESUMEN
During angiogenesis capillary endothelial cells undergo a coordinated set of modifications in their interactions with extracellular matrix components. In this study we have investigated the effect of the prototypical angiogenic factor basic fibroblast growth factor (bFGF) on the expression and function of several integrins in microvascular endothelial cells. Immunoprecipitation experiments with antibodies to individual subunits indicated that microvascular cells express at their surface several integrins. These include the alpha 1 beta 1, alpha 2 beta 1, and alpha 3 beta 1 laminin/collagen receptors; the alpha 6 beta 1 laminin receptor; the alpha 5 beta 1 and alpha v beta 1 fibronectin receptors; the alpha 6 beta 4 basement membrane receptor; and the alpha v beta 3 and alpha v beta 5 vitronectin receptors. Treatment with bFGF caused a significant increase in the surface expression of the alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1, alpha 6 beta 4, and alpha v beta 5 integrins. In contrast, the level of expression of the alpha 1 beta 1 and alpha v beta 3 integrins was decreased in bFGF-treated cells. Immunoprecipitation of metabolically labeled cells indicated that bFGF increases the biosynthesis of the alpha 3, alpha 5, alpha 6, beta 4, and beta 5 subunits and decreases the production of the alpha v and beta 3 subunits. These results suggest that bFGF modulates integrin expression by altering the biosynthesis of individual alpha or beta subunits. In accordance with the upregulation of several integrins observed in bFGF-treated cells, these cells adhered better to fibronectin, laminin, vitronectin, and type I collagen than did untreated cells. The largest differences in beta 1 integrin expression occurred approximately 72 h after exposure to bFGF, at a time when the expression of the endothelial cell-to-cell adhesion molecule endoCAM was also significantly upregulated. In contrast, a shorter exposure to bFGF (24-48 h) was required for the maximal induction of plasminogen activator production in the same cells. Taken together, these results show that bFGF causes significant changes in the level of expression and function of several integrins in microvascular endothelial cells.