RESUMEN
In recent years, classification of soft-tissue sarcomas (STS) has improved with cytogenetic analyses, but their clinical behavior is still not easily predictable. The aim of this study was to detect alterations in the urokinase-type plasminogen system, involved in tumor growth and invasion, by comparing mRNA levels of its components with those of paired normal tissues, and relating them with patient clinical course. Real-time PCR was performed on human STS cell lines and tissues from highly malignant STS, including leiomyosarcomas and malignant fibrous histiocytomas, to evaluate the expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1). Immunohistochemistry of gene products was also performed. Median mRNA values of all genes studied were higher in tumors than in paired normal tissues. In agreement with data on STS cell lines, significant up-regulation for uPA and PAI-1 genes compared to reference values was seen. Moreover, different levels of expression were related to histotype and metastatic phenotype. There was accordance between uPA mRNA and protein expression, while immunodetection of PAI-1 product was weak and scattered. Clearly, the controversial role of PAI-1 protein requires further biological analyses, but evident involvement of uPA/PAI-1 gene overexpression in STS malignancy may highlight a molecular defect useful in discriminating STS high-risk patients.
Asunto(s)
Expresión Génica , Inhibidor 1 de Activador Plasminogénico/metabolismo , Sarcoma/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Adulto , Anciano , Estudios de Casos y Controles , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Supervivencia sin Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Inhibidor 1 de Activador Plasminogénico/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Factores de Riesgo , Sarcoma/clasificación , Sarcoma/diagnóstico , Sarcoma/genética , Sarcoma/patología , Sarcoma/radioterapia , Sarcoma/cirugía , Factores de Tiempo , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
The antimetastatic ruthenium complex imidazolium trans-imidazoledimethylsulfoxide-tetrachlorouthenate (NAMI-A) is tested in the B16 melanoma model in vitro and in vivo. Treatment of B6D2F1 mice carrying intra-footpad B16 melanoma with 35 mg/kg/day NAMI-A for 6 days reduces metastasis weight independently of whether NAMI-A is given before or after surgical removal of the primary tumor. Metastasis reduction is unrelated to NAMI-A concentration, which is 10-fold lower than on primary site (1 versus 0.1 mM), and is correlated to the reduction of plasma gelatinolitic activity and to the decrease of cells expressing CD44, CD54, and integrin-beta(3) adhesion molecules. Metastatic cells also show the reduction of the S-phase cells with accumulation in the G(0)/G(1) phase. In vitro, on the highly metastatic B16F10 cell line, NAMI-A reduces cell Matrigel invasion and its ability to cross a layer of endothelial cells after short exposure (1 h) to 1 to 100 microM concentrations. In these conditions, NAMI-A reduces the gelatinase activity of tumor cells, and it also increases cell adhesion to poly-L-lysine and, in particular, to fibronectin, and this effect is associated to the increase of F-actin condensation. This work shows the selective effectiveness of NAMI-A on the metastatic melanoma and suggests that metastasis inhibition is due to the negative modulation of tumor cell invasion processes, a mechanism in which the reduction of the gelatinolitic activity of tumor cells plays a crucial role.
Asunto(s)
Antineoplásicos/uso terapéutico , Dimetilsulfóxido/análogos & derivados , Neoplasias Pulmonares/tratamiento farmacológico , Melanoma Experimental/tratamiento farmacológico , Compuestos Organometálicos/uso terapéutico , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Moléculas de Adhesión Celular/biosíntesis , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Dimetilsulfóxido/uso terapéutico , Regulación hacia Abajo , Femenino , Pie , Neoplasias Pulmonares/secundario , Melanoma Experimental/secundario , Ratones , Ratones Endogámicos , Invasividad Neoplásica , Compuestos de Rutenio , Neoplasias Cutáneas/patologíaRESUMEN
Imidazolium trans-imidazoledimethylsulphoxidetrachlororuthenate (NAMI-A) was tested in vitro on the pro-adhesive properties, evaluated as resistance to trypsin treatment, which is a bona fide measure of adhesion strength, of KB and HeLa carcinoma cell lines and on human polymorphonuclear neutrophils (HPMN). NAMI-A increased the pro-adhesive activity of KB cells at 0.001 mM concentration, after few minutes incubation and this effect was not influenced by the vehicle used for cell challenge, neither did it depend on NAMI-A concentration or on temperature. The same effect occurred on HeLa cells at 0.01 mM NAMI-A. This effect, detected at concentrations up to 100 times lower than those necessary to block cells at the G(2)-M premitotic phase of cell cycle, or to inhibit matrix metalloproteinase release or cell invasion, was not related to ruthenium uptake by tumour cells. HeLa cells and healthy HPMN, following short exposure to 0.1 mM NAMI-A, assumed a different shape, with the extrusion of filopodia (HeLa) and of large lamellopodia (HPMN), which increased their interactions with the substrate. This effect was attributed to stabilisation, altered turnover and sensitivity to cytochalasin D of actin filaments. Provided that adhesion is associated with cell motility and invasion, these data suggest that NAMI-A may exert antimetastatic properties at concentrations lower than those observed in the lungs at the end of a conventional intraperitoneal treatment in vivo.
Asunto(s)
Antineoplásicos/farmacología , Dimetilsulfóxido/análogos & derivados , Dimetilsulfóxido/farmacología , Neoplasias/tratamiento farmacológico , Neutrófilos/efectos de los fármacos , Compuestos Organometálicos/farmacología , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/ultraestructura , Anticuerpos Bloqueadores/farmacología , Antineoplásicos/análisis , Adhesión Celular/efectos de los fármacos , Línea Celular , Dimetilsulfóxido/análisis , Células HeLa , Humanos , Integrinas/inmunología , Microscopía Confocal , Microscopía Electrónica de Rastreo , Neoplasias/patología , Neutrófilos/química , Neutrófilos/ultraestructura , Compuestos Organometálicos/análisis , Rutenio/análisis , Compuestos de Rutenio , TripsinaRESUMEN
To determine whether subendothelial laminins (LNs) could be implicated in the extravasation of neoplastic lymphocytes, we have examined the distribution of a number of LN isoforms in human vascular structures of adult individuals and have assayed the ability of the isolated LN molecules to promote adhesion of lymphoma and leukemic cells in vitro using a novel cell adhesion assay, CAFCA, Centrifugal Assay for Fluorescence-based Cell Adhesion (E. Giacomello et al., Biotechniques, 26: 758-762, 1999; P. Spessotto et al., Methods Mol. Biol., 139: 321-343, 2000). The use of previously characterized LN chain-specific antibodies showed that the vast majority of the smaller vascular compartments, known to correspond to sites of lymphocyte transmigration, expressed the subunits involved in the structuring of 9 of the 12 LN isoforms known to date. Eight LN isoforms (i.e., LN-1, -2, -4, -5, -8, -9, -10, and -11) and four naturally occurring LN complexes were isolated from various tissues and cultured cells by combined gel filtration, ion exchange, and immunoaffinity chromatographies, and the identity/composition of the isolated LNs/LN complexes was asserted by immunochemical means and amino-acid sequencing. Notwithstanding the widespread colocalization of LN isoforms, a panel of neoplastic B- and T-cell lines and lymphocytes isolated from patients affected by chronic lymphocytic B-cell leukemia attached preferentially and with high avidity to purified LN-8, purified LN-10, and LN-10-containing protein complexes, whereas lymphocytes derived from patients diagnosed with acute lymphocytic leukemia failed to bind to these LNs. All of the tested neoplastic lymphocytes failed to adhere to the isolated LN-1, LN-4, LN-9, and LN-11 and attached moderately well to purified LN-2 and LN-5. The interaction of transformed lymphocytes with LNs was cation-dependent and interchangeably mediated by the alpha3beta1 and alpha6beta1 integrins. The degree of engagement of the two LN receptors was dependent upon their relative levels of cell surface expression, whereas, irrespective of the phenotype, lymphocytes deprived of either of these receptors were incapable of LN binding. The findings suggest that LN-8 and LN-10 may act in an independent or complementary fashion as primary components of the endothelial basement membrane favoring the interaction of extravasating neoplastic lymphocytes. Thus, our results would demonstrate that different LN isoforms may evoke diverse cellular responses in different cell types and that this divergence may be the basis for the redundancy of LN distribution in a number of vascular structures.
Asunto(s)
Linfocitos B/patología , Endotelio Vascular/metabolismo , Laminina/metabolismo , Linfocitos T/patología , Animales , Anticuerpos/farmacología , Anticuerpos Monoclonales , Linfocitos B/metabolismo , Membrana Basal/metabolismo , Bovinos , Adhesión Celular/fisiología , Endotelio Vascular/citología , Humanos , Integrinas/antagonistas & inhibidores , Integrinas/inmunología , Integrinas/metabolismo , Laminina/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Leucemia de Células T/patología , Linfoma de Células T/patología , Linfocitos T/metabolismoRESUMEN
The EMILINs are a new family of glycoproteins of the extracellular matrix. The prototype of this family is the chicken EMILIN that was originally identified in extracts of aortas; it was then found to be widely distributed in several tissues associated with elastin and localized at the interface between amorphous elastin and microfibrils. Based on peptide sequences, chicken and human cDNAs coding for EMILIN were isolated by RT/PCR by screening kidney and heart cDNA libraries. By using a C-terminal fragment of human EMILIN-1 as a bait in the yeast two-hybrid system, a second family member, EMILIN-2, has also been isolated. EMILINs are characterized by a C-terminal gC1q globular domain, a short collagenous sequence, a long coiled-coil region and a new cysteine-rich N-terminal domain that can be considered a hallmark of the family being present also in multimerin. The gene for EMILIN-1 was mapped on chromosome 2p23 overlapping with the promoter region of the ketohexokinase gene. The gC1q domain of EMILIN-1 can form relatively stable and compact homotrimers and this association is then followed by a multimeric assembly of disulfide-bonded protomers. Recombinant EMILIN-1 purified from the supernatant of 293 cells represents a very efficient ligand for cell adhesion of several cell types.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Proteínas de la Matriz Extracelular/fisiología , Glicoproteínas de Membrana/fisiología , Animales , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Movimiento Celular/fisiología , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismoAsunto(s)
Movimiento Celular/fisiología , Animales , Adhesión Celular/fisiología , Fluorescencia , HumanosRESUMEN
The growth capacity and adaptation of TS/A and TS/A-IL4 lines on laminin, fibronectin, collagens I and IV and matrigel compared to plastics were studied by flow cytometry. On plastic plates, TS/A-IL4 grows in vitro more slowly than the TS/A line and shows a more differentiated phenotype. TS/A-IL4 cells loose the capacity to bind lymphocytes and peroxidase positive cells obtained from mice implanted with the same tumour. The ratio between fibroblast- and epithelial-like cells of TS/A adenocarcinoma is subjected to marked modifications depending on the substrate on which the two cell lines are grown. IL4 release per cell unit is increased by collagen I as is the number of CD54 positive cells, suggesting that, at least in part, the in vivo rejection of TS/A-IL4 tumor might be ascribed to the stimulatory effect of the tissue on the IL4 release by tumor cells. The overall result is that gene modified TS/A-IL4 line shows marked changes of behaviour, most of them depending on the substrate on which tumor cells are growing.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Interleucina-4/genética , Neoplasias Mamarias Experimentales/patología , Animales , Adhesión Celular , Técnicas de Cultivo de Célula/instrumentación , Ciclo Celular , Diferenciación Celular , Células Clonales/metabolismo , Células Clonales/patología , Técnicas de Cocultivo , Colágeno , Combinación de Medicamentos , Células Epiteliales/patología , Femenino , Fibroblastos/patología , Fibronectinas , Rechazo de Injerto , Molécula 1 de Adhesión Intercelular/análisis , Interleucina-4/metabolismo , Laminina , Activación de Linfocitos , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas de Neoplasias/análisis , Trasplante de Neoplasias , Plásticos , Proteoglicanos , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patologíaRESUMEN
Engagement of cell surface adhesion receptors with extracellular constituents and with cellular counter-receptors is crucial for the extravasation of blood-borne neoplastic cells and their seeding at distant sites; however, the early events of tumor dissemination-ie, the intravasation step(s)-have been largely neglected. A role for the alpha4beta7 integrin was hypothesized to explain the high leukemogenicity exhibited by one (NQ22) among several T-cell lymphomas studied. To clarify the mechanisms of early aggressivity, the behavior of highly and poorly leukemogenic cell lines were compared in vitro. Cocultivation of physically separated leukemic cells with resting endothelial cells resulted in the up-regulation of VCAM-1 expression. NQ22 cells expressed mRNA of different cytokines that up-regulate VCAM-1 and at higher levels than cells of a nonaggressive lymphoma, and they migrated more efficiently through an activated endothelial cell layer. With the use of neutralizing antibodies against interferon-gamma, granulocyte macrophage colony-stimulating factor, and tumor necrosis factor (TNF)-alpha, it was determined that TNF-alpha is one of the soluble factors released by NQ22 cells involved in the up-regulation of VCAM-1. The finding that vascular cells within the early local growth were strongly positive for VCAM-1 indicated that NQ22 cells could activate endothelial cells also in vivo. Finally, cocultivation of preleukemic alpha4(-)NQ22 cells with TNF-alpha-activated endothelial cells induced the expression of alpha4 integrins on the former cells. Reciprocal up-regulation and engagement of alpha4/VCAM-1 pairs determined the sequential transmigration and intravasation steps, and similar mechanisms might affect the aggressivity of human T lymphoblastic lymphomas.
Asunto(s)
Integrinas/genética , Linfoma de Células T/patología , Invasividad Neoplásica , Molécula 1 de Adhesión Celular Vascular/genética , Animales , Anticuerpos , Adhesión Celular , Movimiento Celular , Técnicas de Cocultivo , Endotelio Vascular/fisiología , Femenino , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Integrinas/fisiología , Interferón gamma/inmunología , Interferón gamma/fisiología , Masculino , Ratones , Ratones Endogámicos AKR , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/fisiología , Molécula 1 de Adhesión Celular Vascular/fisiologíaRESUMEN
Neutrophils and macrophages express on their membrane molecules which may, in principle, interact with each other, promote specific cell to cell adhesion, affect cell function and finally, as a consequence, modulate the progression of the inflammatory process. We tested therefore if human neutrophils specifically adhere to human monocyte-derived macrophage monolayer (MDMM). Our findings show that neutrophils significantly adhere to 4-day old MDMM and that the extent of adhesion is increased by LPS-activation of MDMM. The specificity of the interaction was shown by the very low extent of adhesion of neutrophils either to freshly prepared monocyte or other types of cell monolayers and by the low percent of adhesion showed by eosinophils exposed to 7-day old MDMM. A role for beta2 integrins, CD31 and PAF-receptor in the mechanism of neutrophil-MDMM interaction is suggested by specific antagonists. We suggest that the adhesion between the two cell types could lead to an increase in concentration of neutrophil- or macrophage released factors in the interaction site and in a mutual modulation of phagocyte functions.
Asunto(s)
Macrófagos/fisiología , Monocitos/citología , Neutrófilos/fisiología , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , Antígenos CD18/fisiología , Cationes Bivalentes/farmacología , Adhesión Celular/fisiología , Células Cultivadas , Medios de Cultivo/química , Humanos , Molécula 1 de Adhesión Intercelular/fisiología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/ultraestructura , Neutrófilos/ultraestructura , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/fisiología , Glicoproteínas de Membrana Plaquetaria/fisiologíaRESUMEN
The Ref-1 (also called APE or HAP1) protein is a bifunctional enzyme impacting on a wide variety of important cellular functions. It acts as a major member of the DNA base excision repair pathway. Moreover, Ref-1 stimulates the DNA-binding activity of several transcription factors (TFs) through the reduction of highly reactive cysteine residues. Therefore, it represents a mechanism that regulates eukaryotic gene expression in a fast way. However, it has been demonstrated that external stimuli directly act on Ref-1 by increasing its expression levels, a time-consuming mechanism representing a paradox in terms of rapidity of TF regulation. In this paper we demonstrate that this is only an apparent paradox. Exposure of B lymphocytes to H(2)O(2)induced a rapid and sustained increase in Ref-1 protein levels in the nucleus as evaluated by both western blot analysis and by pulse-chase experiments. A time course, two color in situ immunocytochemistry indicated that the up-regulation of Ref-1 in the nucleus at <30 min was primarily the consequence of translocation of its cytoplasmic form. This early nuclear accumulation is effective in modulating the DNA-binding activity of the B cell-specific activator protein BSAP/Pax-5. In fact, EMSA experiments demonstrate that a transient interaction with Ref-1 up-regulates the DNA-binding activity of BSAP/Pax-5. Moreover, in a co-transfection experiment, Ref-1 increased the BSAP/Pax-5 activating effect on an oligomerized BSAP/Pax-5 binding site of the CD19 promoter by 5- to 8-fold. Thus, Ref-1 mediates its effect by up-regulating the DNA-binding activity of BSAP/Pax-5, accounting for a new and fast outside/inside pathway of signaling in B cells.
Asunto(s)
Linfocitos B/fisiología , Liasas de Carbono-Oxígeno/fisiología , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Proteínas de Unión al ADN/fisiología , Proteínas Nucleares/fisiología , Transducción de Señal/fisiología , Transporte Biológico/fisiología , Línea Celular , Humanos , Oxidación-Reducción , Factor de Transcripción PAX5 , Factores de Transcripción/fisiologíaRESUMEN
The in vitro/in vivo growth capacity and phenotype of TS/A and the IL4-transfected TS/A-IL4 cell lines were studied by cell cycle analysis, expression of ICAM-1/CD54, transferrin receptor/CD71 and E-cadherin and by histology of the primary tumors. TS/A-IL4, unlike the TS/A line, shows in vitro a marked increase in the fibroblastoid cell type and a decreased E-cadherin expression. Administration of conditioned medium containing IL4 obtained from the TS/A-IL4 cell line, stimulates CD54 expression in the TS/A cell line. TS/A-IL4 tumors grow more slowly in vivo and are ultimately rejected. These processes are accompanied by a marked increase in collagen and extracellular matrix proteins and increased recruitment and degranulation of mast cells. The paracrine effect of IL4, released by the transfected tumor cells, might be responsible for the reduced in vivo growth of the TS/A cell line in the presence of TS/A-IL4 cells.
Asunto(s)
Adenocarcinoma/terapia , Interleucina-4/genética , Neoplasias Mamarias Animales/terapia , Adenocarcinoma/patología , Animales , Ciclo Celular , División Celular , Femenino , Terapia Genética , Interleucina-4/uso terapéutico , Neoplasias Mamarias Animales/patología , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Comunicación Paracrina , Fenotipo , Transfección , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
We have identified type VI collagen (Col VI) as a primary subendothelial extracellular matrix component responsible for von Willebrand factor (vWF)-dependent platelet adhesion and aggregation under high tensile strength. Intact tetrameric Col VI was the form of the collagen found to be capable of promoting vWF-mediated platelet adhesion/aggregation under this shear condition, whereas removal of the predominant portion of the terminal globules by pepsin treatment abrogated its activity. The inability of the pepsin-digested Col VI to support any platelet interaction at high flow was because of the failure of the A3(vWF) domain to bind to this form of collagen, suggesting a stringent requirement of a tridimensional conformation or of intactness of its macromolecular structure. In contrast, the A1(vWF) domain bound to both intact and pepsin-digested Col VI tetramers but, in accordance with the cooperating function of the two vWF domains, failed to support platelet adhesion/aggregation under high shear onto Col VI by itself. The putative A1(vWF) binding site resided within the A7(VI) module (residues 413-613) of the globular amino-terminal portion of the alpha3(VI) chain. Soluble recombinant A7(VI) polypeptide strongly perturbed the vWF-mediated platelet adhesion to Col VI under high shear rates, without affecting the binding of the vWF platelet receptor glycoprotein Ibalpha to its cognate ligand A1(vWF). The findings provide evidence for a concerted action of the A1(vWF) and A3(vWF) domains in inducing platelet arrest on Col VI. This is accomplished via an interaction of the A1(vWF) domain with a site contained in the alpha3 chain A7(VI) domain and via a conformation-dependent interaction of the A3(vWF) domain with the intact tetrameric collagen. The data further emphasize that Col VI microfilaments linking the subendothelial basement membrane to the interstitial collagenous network may play a pivotal role in the hemostatic process triggered upon damage of the blood vessel wall.
Asunto(s)
Colágeno/metabolismo , Adhesividad Plaquetaria , Factor de von Willebrand/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Membrana Basal/metabolismo , Sitios de Unión , Plaquetas/metabolismo , Colágeno/inmunología , Humanos , Integrinas/metabolismo , Ratones , Pepsina A/metabolismo , Conformación Proteica , Receptores de Colágeno , Proteínas Recombinantes/metabolismo , Ristocetina/metabolismoRESUMEN
Inhibition of growth or eradication of experimentally induced tumors has been shown to be accompanied by infiltration of eosinophils and macrophages into the tumor mass. Since macrophages are important mediators of host antitumor activity, the possibility arises that a collaboration may exist between these two cell types in the control of tumor growth. In this study, we report the effect of eosinophil peroxidase (EPO), a basic protein contained in eosinophils that binds to several cell types including macrophages, on tumor necrosis factor (TNF) production and hydrogen peroxide release by human monocyte-derived macrophages. After incubation with EPO, the macrophages produced large amounts of TNF and displayed an enhanced phorbol 12-myristate 13-acetate-triggered hydrogen peroxide release. These effects were accompanied by an increased cell protein content and by morphologic changes leading the large, round macrophages of the control cultures to become elongated, pear-like or spindle shaped cells after treatment with EPO. The stimulatory effect of EPO on hydrogen peroxide release was insensitive to addition of exogenous catalase, a H2O2-degrading enzyme, suggesting that an extracellular catalytic activity of EPO was not involved. In addition, myeloperoxidase, the homologous peroxidase of neutrophils with a catalytic activity similar to that of EPO, was ineffective. The EPO-induced effects differed in several aspects from the effects of lipopolysaccaride and interferon-gamma, two well-known macrophage activators. These findings provide supportive evidence for a functional interrelationship between eosinophils and macrophages that may be physiologically relevant in the tumoricidal activity of macrophages.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Peroxidasas/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Catalasa/farmacología , Tamaño de la Célula , Células Cultivadas , Medios de Cultivo Condicionados , Peroxidasa del Eosinófilo , Humanos , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Linfoma de Células B/patología , Macrófagos/metabolismo , Macrófagos/ultraestructura , Ratones , Monocitos/citología , Peroxidasa/farmacología , Polimixina B/farmacología , Biosíntesis de Proteínas , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
The effect of Factor AF2 (AF2), a standardized fraction of peptides with a molecular weight of < 10,000 Dalton obtained from livers and spleens of newborn lambs, on the differentiation of human monocyte-derived macrophages was studied, in view of the central role played by these cells in inflammation and tumor cytotoxicity. The results show that the drug 1. increases the cell density of cultures, 2. favours the morphologic differentiation of monocytes into macrophages, and 3. increases the macrophages phagocytic capacity. The first two effects are observed when monocytes are cultured in 1% serum but not in 10% serum while the enhancement of phagocytic activity is detected at both serum concentrations.
Asunto(s)
Inductores de Interferón/farmacología , Hígado/química , Macrófagos/efectos de los fármacos , Péptidos/farmacología , Bazo/química , Extractos de Tejidos/farmacología , Animales , Animales Recién Nacidos/fisiología , Diferenciación Celular/efectos de los fármacos , Humanos , Técnicas In Vitro , Indicadores y Reactivos , Inductores de Interferón/aislamiento & purificación , Macrófagos/ultraestructura , Péptidos/aislamiento & purificación , Fagocitosis/efectos de los fármacos , Ovinos , Extractos de Tejidos/aislamiento & purificaciónRESUMEN
Solcoseryl is a protein-free, standardized dialysate/ultrafiltrate derived from calf blood, which has been shown to improve situations of impaired healing in both experimental animals and man. Its activity seems to be multifactorial although the precise cellular and molecular mechanisms contributing to its effect have not been fully elucidated. Since monocyte-derived macrophages play a central role in inflammation and particularly in wound healing and tissue remodelling, the effect of the dialysate on human monocytes cultured in vitro for 10 days in the presence of human serum was studied. The results show that the drug, at concentrations ranging from 0.2 to 2%, increases the cell density of the cultures and the cell protein content, and favours the differentiation of monocytes into macrophages when 1% serum concentration is used in the culture medium. These effects are no more apparent when the serum concentration was raised to 10%. These data suggest that the drug may substitute, at least in part, for serum in monocyte-macrophage cultures. The observed effects give a sound basis for at least a partial explanation of the therapeutic effects of the drug, particularly at sites where the supply of serum-derived factors is limited.
Asunto(s)
Actiemil/farmacología , Monocitos/efectos de los fármacos , Animales , Proteínas Sanguíneas/metabolismo , Bovinos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Fibroblastos/efectos de los fármacos , Humanos , Indicadores y Reactivos , Macrófagos/efectos de los fármacosRESUMEN
The effect of Factor AF2 (AF2), a xenogeneic fraction of peptides with a molecular weight of < 10,000 Dalton obtained from livers and spleens of newborn lambs, on the oxygen consumption and the phagocytic activity of human polymorphonuclear leukocytes (PMN) was studied. AF2 increased the oxygen uptake of PMN exposed both to serum-treated zymosan (STZ), a phagocytosable stimulus, and phorbol-myristate-acetate (PMA), a soluble stimulus. The potentiating effect of the drug was dose-dependent and more pronounced when suboptimal amounts of either stimulus were used. The phagocytic activity of PMN, as measured by the rate of mineral oil particles ingestion, was also increased by AF2 in a dose-dependent manner. These results suggest that the drug may influence PMN behaviour in at least two ways: 1. by increasing the rate of phagocytosis, and 2. by potentiating the respiratory burst induced by soluble and particulate stimuli. The results are discussed in relation to the beneficial effects of AF2 in cancer patients under chemotherapy or radiation treatment.
Asunto(s)
Hígado/química , Neutrófilos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Estallido Respiratorio/efectos de los fármacos , Bazo/química , Extractos de Tejidos/farmacología , Adulto , Animales , Femenino , Humanos , Técnicas In Vitro , Cinética , Masculino , Persona de Mediana Edad , Oxidación-Reducción , Consumo de Oxígeno/efectos de los fármacos , Ovinos , Zimosan/farmacologíaRESUMEN
Five eosinophil peroxidase (EPO)-deficient subjects were identified from 131,000 peripheral blood samples examined for routine automated analysis. The EPO-deficient eosinophils of these subjects met the main criteria established for EPO deficiency: absent or strongly decreased reaction for peroxidase, absent or strongly decreased staining with Sudan Black, and an increased ratio of the granule core volume to the total granule volume. In this report we show that this granule alteration is caused mainly by a decrease of its volume, particularly of the matrix, and that two other matrix proteins, eosinophil cationic protein and eosinophil derived neurotoxin, appear to be present in normal amounts in the EPO-deficient granules.
Asunto(s)
Gránulos Citoplasmáticos/enzimología , Eosinófilos/enzimología , Peroxidasas/deficiencia , Adulto , Anciano , Gránulos Citoplasmáticos/ultraestructura , Peroxidasa del Eosinófilo , Eosinófilos/patología , Eosinófilos/ultraestructura , Femenino , Humanos , Masculino , Microscopía Electrónica , Microscopía Inmunoelectrónica , Persona de Mediana Edad , Valores de ReferenciaRESUMEN
Although an elastolytic activity in eosinophil-rich cell fractions from mice has been reported, this enzyme has not been purified and characterized as yet in any mammalian species. Eosinophilic elastase was isolated from human eosinophil fragments (cytosomes) obtained from normal and eosinophilic subjects. The enzyme was purified to apparent electrophoretic homogeneity by fast protein liquid chromatography. The enzyme shows the same physical properties of the major elastase isoenzyme of human neutrophils. In addition, like monocyte elastase, it reacts with a monoclonal antibody against human neutrophil elastase. The biochemical similarities observed between the above-mentioned enzymes and the immunolocalization findings strongly support the idea that human eosinophils and neutrophils contain the same enzyme activity. Eosinophils show immunoreactive material in both types of dense cytoplasmic granules. This observation supports the current hypothesis that the different types of eosinophilic granules represent successive morphological stages of maturation.
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Eosinófilos/enzimología , Elastasa Pancreática/química , Reacciones Cruzadas , Eosinófilos/química , Eosinófilos/ultraestructura , Humanos , Immunoblotting , Inmunohistoquímica , Cinética , Mieloblastina , Neutrófilos/química , Elastasa Pancreática/inmunología , Elastasa Pancreática/aislamiento & purificación , Serina Endopeptidasas/química , Fracciones Subcelulares/química , Fracciones Subcelulares/enzimologíaRESUMEN
Production of O2- in response to FMLP, TNF, IFN-gamma, platelet activating factor, LPS, substance P, and PMA by human eosinophils in suspension and in contact with polystyrene ELISA plastic (PL) or biologic surfaces was studied. Monolayers of human endothelial cells (HEC) or PL coated with FCS, fibronectin, laminin, collagen types I and IV, fibrinogen, or fibrin were used as biologic surfaces. Only PMA and FMLP stimulated O2- generation by eosinophils in suspension. Eosinophils residing on HEC monolayers, either untreated or treated with LPS, were unresponsive to all stimuli except PMA. PMA induced O2- generation by eosinophils on all surfaces; FMLP on all surfaces but HEC monolayers; TNF and platelet-activating factor only on PL, fibrinogen, and fibrin; LPS and substance P only on PL. PMA was equally effective on eosinophils on surfaces and in suspension, whereas the effect of FMLP was greater on eosinophils on surfaces than on eosinophils in suspension. IFN-gamma was ineffective on any of the surfaces tested. These results indicate that biologic surfaces may profoundly affect the ability of eosinophils to respond with a respiratory burst to physiologically relevant soluble stimuli, the effect varying according to the nature of both the stimulus and the surface. Since the respiratory burst generates products of oxygen reduction that are toxic to several tissue components, it follows that biologic surfaces may modulate eosinophil-induced tissue injury.
Asunto(s)
Endotelio Vascular/fisiología , Eosinófilos/fisiología , Matriz Extracelular/fisiología , Superóxidos/metabolismo , Eosinófilos/metabolismo , Humanos , Técnicas In Vitro , Inflamación/fisiopatología , Lipopolisacáridos/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Factor de Activación Plaquetaria/farmacología , Sustancia P/farmacología , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The production of O2- in response to LPS, PAF, FMLP, TNF and PMA by human neutrophils in suspension and residing on surfaces coated with fetal calf serum (FCS), fibronectin (FN), laminin (LM), collagen types I and IV (CI and CIV), fibrinogen (FBG) or fibrin (FBN) was studied. Of the agonists used, PAF and LPS failed to induce a response in any of the above conditions; FMLP and PMA stimulated neutrophils to produce similar amounts of O2- either in suspension or on biological surfaces; TNF induced O2- production only by cells residing on FN, FBG and FBN. These results indicate that production of oxygen-derived free radicals by neutrophils depends on the type of agonist and the nature of the surface they interact with. The relationship between the respiratory burst and adherence was studied by measuring O2- release and adherence of neutrophils residing on FN, LM, CIV, and FBG, in the absence and in the presence of the monoclonal antibody 60.3 that recognizes the common beta-chain of CD11/CD18 integrins. FMLP, PMA and TNF increased neutrophil adherence on all these surfaces except CIV. The monoclonal antibody markedly inhibited the FMLP and PMA-induced adherence but had no effect on the O2- release elicited by these two agonists. In contrast, the monoclonal antibody inhibited both the increased adherence and O2- release induced by TNF on FN and FBG. The TNF-induced increase in adherence to LM, that was not accompanied by an increase in O2- release, was also inhibited by the monoclonal antibody. We conclude that the respiratory burst of neutrophils residing on surfaces is not necessarily correlated with adherence.