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1.
Nat Genet ; 22(1): 69-73, 1999 May.
Artículo en Inglés | MEDLINE | ID: mdl-10319864

RESUMEN

Craniofacial malformations are among the most frequent congenital birth defects in humans; cleft palate, that is inadequate fusion of the palatal shelves, occurs with an annual incidence of 1 in 700 to 1 in 1,000 live births among individuals of European descent. The secondary palate arises as bilateral outgrowths from the maxillary processes, and its formation depends on the coordinated development of craniofacial structures including the Meckel's cartilage and the mandible. Cleft lip and palate syndromes in humans are associated with polymorphisms in the gene (TGFA) encoding transforming growth factor-alpha (TGF-alpha), an epidermal growth factor receptor (EGFR) ligand made by most epithelia. Here we have characterized craniofacial development in Egfr-deficient (Egfr-/-) mice. Newborn Egfr-/- mice have facial mediolateral defects including narrow, elongated snouts, underdeveloped lower jaw and a high incidence of cleft palate. Palatal shelf explants from Egfr-/- mice fused, but frequently had residual epithelium in the midline. In addition, morphogenesis of Meckel's cartilage was deficient in cultured mandibular processes from Egfr-/- embryos. The secretion of matrix metalloproteinases (MMPs) was diminished in Egfr-/- explants, consistent with the ability of EGF to increase MMP secretion and with the decreased MMP expression caused by inhibition of Egfr signalling in wild-type explants. Accordingly, inactivation of MMPs in wild-type explants phenocopied the defective morphology of Meckel's cartilage seen in Egfr-/- explants. Our results indicate that EGFR signalling is necessary for normal craniofacial development and that its role is mediated in part by its downstream targets, the MMPs, and may explain the genetic correlation of human cleft palate with polymorphisms in TGFA.


Asunto(s)
Receptores ErbB/fisiología , Desarrollo Maxilofacial/fisiología , Hueso Paladar/crecimiento & desarrollo , Cráneo/crecimiento & desarrollo , Animales , Animales Recién Nacidos , Fisura del Paladar/genética , Fisura del Paladar/patología , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/patología , Técnicas de Cultivo , Receptores ErbB/genética , Femenino , Gelatinasas/efectos de los fármacos , Gelatinasas/metabolismo , Mandíbula/efectos de los fármacos , Mandíbula/embriología , Mandíbula/crecimiento & desarrollo , Metaloendopeptidasas/efectos de los fármacos , Metaloendopeptidasas/metabolismo , Ratones , Mutación , Hueso Paladar/embriología , Embarazo , Inhibidores de Proteasas/farmacología , Cráneo/embriología
2.
Curr Opin Cell Biol ; 4(5): 772-81, 1992 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-1329869

RESUMEN

Adhesion receptors allow cells to interact with a dynamic and information-rich environment of extracellular matrix molecules. The integrin family of adhesion receptors transduces signals from the extracellular matrix that regulate growth, gene expression and differentiation, as well as cell shape, motility and cytoskeletal architecture. Recent data support the hypothesis that integrins transduce signals cooperatively with other classes of adhesion receptors or with growth factor receptors. Furthermore, the ability of integrins to interact with the cytoskeleton appears to be fundamental to their mechanism for signal transduction.


Asunto(s)
Matriz Extracelular/metabolismo , Integrinas/metabolismo , Transducción de Señal , Animales , Humanos , Integrinas/química , Receptores de Superficie Celular/metabolismo
3.
Curr Opin Cell Biol ; 8(5): 731-8, 1996 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-8939664

RESUMEN

Cells respond to changes in their microenvironment by altering their cell surface and extracellular matrix proteins. Rapid and irreversible changes in these proteins are possible through their degradation or activation by proteolysis. By focalizing the proteolytic events at or near the cell surface, these processes can be effective even in the presence of high concentrations of inhibitors. Evidence is emerging that secreted and transmembrane matrix metalloproteinases, metalloproteinases of the adamalysin and astacin (tolloid) families, and serine proteinases are crucial in development, differentiation, cell motility and invasion, and cell-extracellular decisions.


Asunto(s)
Endopeptidasas/metabolismo , Matriz Extracelular/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Apoptosis , Membrana Celular/metabolismo , Endopeptidasas/genética , Epitelio/metabolismo , Humanos , Mesodermo , Receptores de Superficie Celular/metabolismo , Transformación Genética
4.
Nat Cell Biol ; 3(3): 267-75, 2001 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-11231576

RESUMEN

Here we show that plasma kallikrein (PKal) mediates a plasminogen (Plg) cascade in adipocyte differentiation. Ecotin, an inhibitor of serine proteases, inhibits cell-shape change, adipocyte-specific gene expression, and lipid accumulation during adipogenesis in culture. Deficiency of Plg, but not of urokinase or tissue-type plasminogen activator, suppresses adipogenesis during differentiation of 3T3-L1 cells and mammary-gland involution. PKal, which is inhibited by ecotin, is required for adipose conversion, Plg activation and 3T3-L1 differentiation. Human plasma lacking PKal does not support differentiation of 3T3-L1 cells. PKal is therefore a physiological regulator that acts in the Plg cascade during adipogenesis. We propose that the Plg cascade fosters adipocyte differentiation by degradation of the fibronectin-rich preadipocyte stromal matrix.


Asunto(s)
Adipocitos/citología , Diferenciación Celular/fisiología , Coagulantes/metabolismo , Proteínas de Escherichia coli , Proteínas Periplasmáticas , Calicreína Plasmática/metabolismo , Plasminógeno/metabolismo , Adipocitos/fisiología , Animales , Compuestos Azo/metabolismo , Proteínas Bacterianas/farmacología , Western Blotting , Células Cultivadas , Colorantes/metabolismo , Medio de Cultivo Libre de Suero , Femenino , Fibrinolisina/metabolismo , Fibronectinas/metabolismo , Humanos , Inmunohistoquímica , Glándulas Mamarias Animales/anatomía & histología , Glándulas Mamarias Animales/citología , Ratones , Inhibidores de Serina Proteinasa/farmacología
5.
Nat Cell Biol ; 2(10): 737-44, 2000 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-11025665

RESUMEN

During carcinogenesis of pancreatic islets in transgenic mice, an angiogenic switch activates the quiescent vasculature. Paradoxically, vascular endothelial growth factor (VEGF) and its receptors are expressed constitutively. Nevertheless, a synthetic inhibitor (SU5416) of VEGF signalling impairs angiogenic switching and tumour growth. Two metalloproteinases, MMP-2/gelatinase-A and MMP-9/gelatinase-B, are upregulated in angiogenic lesions. MMP-9 can render normal islets angiogenic, releasing VEGF. MMP inhibitors reduce angiogenic switching, and tumour number and growth, as does genetic ablation of MMP-9. Absence of MMP-2 does not impair induction of angiogenesis, but retards tumour growth, whereas lack of urokinase has no effect. Our results show that MMP-9 is a component of the angiogenic switch.


Asunto(s)
Transformación Celular Neoplásica , Islotes Pancreáticos/patología , Metaloproteinasa 9 de la Matriz/metabolismo , Neovascularización Patológica , Neoplasias Pancreáticas/irrigación sanguínea , Acetamidas/farmacología , Animales , Factores de Crecimiento Endotelial/aislamiento & purificación , Genes de Cambio , Linfocinas/aislamiento & purificación , Ratones , Ratones Transgénicos , Proteínas Tirosina Quinasas Receptoras/aislamiento & purificación , Receptores de Factores de Crecimiento/aislamiento & purificación , Receptores de Factores de Crecimiento Endotelial Vascular , Transducción de Señal , Distribución Tisular , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular
6.
Nat Med ; 5(6): 623-8, 1999 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-10371499

RESUMEN

Hypertrophic chondrocytes in the epiphyseal growth plate express the angiogenic protein vascular endothelial growth factor (VEGF). To determine the role of VEGF in endochondral bone formation, we inactivated this factor through the systemic administration of a soluble receptor chimeric protein (Flt-(1-3)-IgG) to 24-day-old mice. Blood vessel invasion was almost completely suppressed, concomitant with impaired trabecular bone formation and expansion of hypertrophic chondrocyte zone. Recruitment and/or differentiation of chondroclasts, which express gelatinase B/matrix metalloproteinase-9, and resorption of terminal chondrocytes decreased. Although proliferation, differentiation and maturation of chondrocytes were apparently normal, resorption was inhibited. Cessation of the anti-VEGF treatment was followed by capillary invasion, restoration of bone growth, resorption of the hypertrophic cartilage and normalization of the growth plate architecture. These findings indicate that VEGF-mediated capillary invasion is an essential signal that regulates growth plate morphogenesis and triggers cartilage remodeling. Thus, VEGF is an essential coordinator of chondrocyte death, chondroclast function, extracellular matrix remodeling, angiogenesis and bone formation in the growth plate.


Asunto(s)
Huesos/fisiología , Cartílago/fisiología , Condrocitos/metabolismo , Factores de Crecimiento Endotelial/fisiología , Linfocinas/fisiología , Neovascularización Fisiológica/fisiología , Osteogénesis/fisiología , Animales , Huesos/anatomía & histología , Cartílago/anatomía & histología , División Celular , Condrocitos/citología , Condrocitos/efectos de los fármacos , Inmunoglobulina G/genética , Inmunoglobulina G/farmacología , Ratones , Ratones Endogámicos C57BL , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/farmacología , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/farmacología , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/farmacología , Factores de Crecimiento Endotelial Vascular
7.
J Exp Med ; 147(6): 1695-1712, 1978 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-210248

RESUMEN

The effects of glucocorticoids on biochemical functions of macrophages from man, mouse, rabbit, and guinea pig were examined. Secretion of plasminogen activator by human peripheral blood monocytes was decreased by 50% with 1 nM dexamethasone. Differentiation of murine monocytic and granulocytic colonies in agar from bone marrow precursors was decreased by 50% at 7 days with 20 nM dexamethasone. Secretion of elastase, collagenase, and plasminogen activator by resident and thioglycollate-elicited mouse peritoneal macrophages was decreased by dexamethasone, cortisol, and triamcinolone acetonide (1--1,000 nM), but not by progesterone, estradiol, and dihydrotestosterone (1,000 nM); in contrast, secretion of lysozyme was not affected by glucocorticoids or other steroids. The inhibition of macrophage secretion by dexamethasone was both time and dose dependent. Effects were detected within 1--6 h after addition of the glucocorticoids, became maximum by 24 h, and were reversed during a similar time period after removal of the hormones. The extent of inhibition of macrophage secretion increased with increasing glucocorticoid concentration. Half-maximum inhibition of secretion of elastase, collagenase, and plasminogen activator was seen at dexamethasone concentrations (1--10 nM) similar to those that half-saturated the specific glucocorticoid receptors in these cells. At high concentrations of dexamethasone (100--1,000 nM) the secretion of plasminogen activator was inhibited to a greater extent (greater than 95%) than the secretion of elastase (60--80%). Progesterone alone had no effect on secretion, but it blocked the inhibitory effects of dexamethasone and cortisol. Secretion of collagenase, neutral proteinases, and plasminogen activator by elicited rabbit alveolar macrophages was inhibited with glucocorticoids (0.1--100 nM) but not with progesterone or sex steroids. Secretion of a neutral elastinolytic proteinase by guinea pig alveolar macrophages was also inhibited by dexamethasone. These data support the regulatory role of glucocorticoids on macrophage functions at physiological concentrations.


Asunto(s)
Glucocorticoides/farmacología , Macrófagos/efectos de los fármacos , Colagenasa Microbiana/metabolismo , Elastasa Pancreática/metabolismo , Activadores Plasminogénicos/metabolismo , Animales , Líquido Ascítico/citología , Médula Ósea/efectos de los fármacos , Células Clonales , Dexametasona/farmacología , Glucocorticoides/antagonistas & inhibidores , Granulocitos/efectos de los fármacos , Cobayas , Macrófagos/enzimología , Ratones , Monocitos/efectos de los fármacos , Progesterona/farmacología , Alveolos Pulmonares/citología , Conejos , Tioglicolatos/inmunología
8.
J Exp Med ; 158(4): 1272-93, 1983 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-6619735

RESUMEN

Macrophages are active secretory cells that display functionally distinct phenotypes that are regulated by inflammation. We have found that apoprotein E (ApoE), a component of plasma lipoproteins, was synthesized and secreted by resident and nonspecifically stimulated macrophages elicited with thioglycollate broth, but not by activated macrophages obtained from mice treated with bacillus Calmette-Guerin, pyran copolymer, whole Corynebacterium parvum, or bacterial endotoxin. ApoE represented approximately 1% of the newly synthesized protein and approximately 10% of secreted protein of resident and thioglycollate-elicited macrophages. ApoE from thioglycollate-elicited macrophages was indistinguishable from ApoE in mouse plasma lipoproteins, as determined by immunoreactivity, peptide mapping, and molecular weight. When specific antibodies were used to localize cell-associated ApoE, strong immunofluorescence was seen in the Golgi region of resident and thioglycollate-elicited macrophages immediately after removal from the peritoneal cavity, as well as after culture for up to 7 d. In contrast, activated macrophages did not synthesize or secrete ApoE to an appreciable extent and had no immunocytochemically detectable intracellular ApoE. When activated macrophages were cultured in medium containing serum, their activated state, as judged by production of H2O2, declined within 48-72 h in parallel with the induction of synthesis and secretion of ApoE and detection of intracellular ApoE by immunofluorescence. During prolonged culture the rate of synthesis and secretion of ApoE increased in both resident and activated macrophages. Therefore, the synthesis and secretion of ApoE may serve as markers for the functional state of macrophages.


Asunto(s)
Apolipoproteínas/biosíntesis , Activación de Macrófagos , Macrófagos/metabolismo , Animales , Apolipoproteínas/metabolismo , Apolipoproteínas E , Vacuna BCG/farmacología , Supervivencia Celular , Células Cultivadas , Terapia de Inmunosupresión , Inflamación/inmunología , Inflamación/patología , Macrófagos/clasificación , Macrófagos/inmunología , Ratones , Ratones Endogámicos C3H , Peso Molecular , Fenotipo , Copolímero del Pirano/farmacología , Conejos , Ratas , Tioglicolatos/farmacología
9.
J Exp Med ; 159(1): 167-78, 1984 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-6198420

RESUMEN

We have determined the effect of various endocytic ligands on the secretion of ApoE by macrophages. ApoE was a major secreted protein of resident macrophages, but BCG-activated macrophages secreted little ApoE and periodate-elicited macrophages secreted intermediate amounts of ApoE. Resident, periodate-elicited, and BCG-activated mouse peritoneal macrophages were incubated with AcLDL, EIgG, EIgMC, dextran sulfate, latex, or zymosan, and the resulting protein secretion patterns were analyzed by [35S]methionine labeling and SDS-polyacrylamide gel electrophoresis. AcLDL increased total [35S]methionine incorporation into secreted proteins. Although AcLDL increased the secretion of ApoE by resident macrophages less than or equal to fivefold in a dose-dependent manner, with maximal stimulation at 4.8 micrograms/ml, it decreased the secretion of ApoE by periodate-elicited macrophages to almost nothing and did not affect the low rate of secretion of ApoE by BCG-activated macrophages. However, EIgG, which increases cellular cholesterol content of macrophages as AcLDL does, did not increase ApoE secretion, and dextran sulfate, which is recognized by the same receptor as AcLDL, also did not increase ApoE secretion. The binding and uptake of EIgG, dextran sulfate, zymosan, latex, and EIgMC all decreased the secretion of ApoE. These endocytic ligands also altered the pattern of secreted and cellular proteins other than ApoE. The pattern of response was ligand-specific. However, increased secretion of polypeptides of Mr 62,000 and 68,000 was common to many stimuli. We conclude that receptor-mediated endocytosis modulates the secretion of ApoE and other proteins pleiotypically in resident, inflammatory, and activated macrophages.


Asunto(s)
Apolipoproteínas/metabolismo , Endocitosis , Macrófagos/metabolismo , Receptores Fc/fisiología , Animales , Apolipoproteínas E , Sulfato de Dextran , Dextranos/farmacología , Inflamación/inmunología , Látex/farmacología , Lipoproteínas LDL/farmacología , Activación de Macrófagos , Macrófagos/clasificación , Macrófagos/inmunología , Ratones , Péptidos/metabolismo , Receptores de IgG , Receptores Inmunológicos/fisiología , Zimosan/farmacología
10.
J Exp Med ; 159(1): 152-66, 1984 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-6229594

RESUMEN

We have determined that the interaction of IgG-coated erythrocytes (EIgG) and complement-coated erythrocytes (EIgMC) with macrophage Fc and complement receptors, respectively, modulates the secretion of the neutral proteinases, elastase, and plasminogen activator. EIgG binding and ingestion stimulated secretion of elastase and plasminogen activator less than or equal to 6-fold and 20-fold, respectively, over the 3 d following treatment. Stimulation was dependent on the IgG titer bound to each erythrocyte and was detectable at greater than 6.2 X 10(3) molecules IgG/ erythrocyte (total 0.99 nM IgG in the culture). Cytochalasin B did not inhibit stimulation, indicating that the ingestion of ligands was not necessary. Binding of EIgG to the three subclass-specific Fc receptors (IgG2a, IgG2b/IgG1, IgG3) was effective. Stimulation of elastase secretion required continued exposure of ligands to cells for up to 24 h, whereas production of plasminogen activator, which has plasma membrane-bound forms as well as secreted forms, was stimulated by exposure for 2 h. The stimulated production of elastase and plasminogen activator by triggering Fc receptors was seen only when the initial secretion rates were low. Periodate- or thioglycollate-elicited macrophages, which have high rates of proteinase secretion, were not stimulated further. EIgMC, which are bound but not ingested by resident macrophages, stimulated elastase secretion transiently, and the rate of secretion returned to the control level by 24 h. Therefore, the mode of stimulation of neutral proteinase secretion by complement receptor differed from that of Fc receptor; stimulation by complement receptor possibly involves a limited release of enzyme from intracellular stores, rather than stimulating accelerated synthesis of enzyme. Erythrocytes coated with both complement and IgG showed both the transient increase in elastase typical of complement-mediated secretion and the sustained increase typical of Fc receptor-mediated secretion. These results suggest that macrophage Fc and complement receptors regulate secretion of proteinases by receptor-specific mechanisms.


Asunto(s)
Macrófagos/enzimología , Elastasa Pancreática/metabolismo , Activadores Plasminogénicos/metabolismo , Receptores Fc/fisiología , Receptores Inmunológicos/fisiología , Animales , Relación Dosis-Respuesta Inmunológica , Eritrocitos/metabolismo , Femenino , Inflamación/inmunología , Activación de Macrófagos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Fagocitosis , Receptores de Complemento/fisiología , Receptores de Complemento 3b , Receptores Fc/clasificación , Receptores de IgG , Receptores Inmunológicos/clasificación , Factores de Tiempo
11.
J Exp Med ; 142(2): 346-60, 1975 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-167095

RESUMEN

Thioglycollate-stimulated mouse macrophages release a specific collagenase into their medium during in vitro culture. The macrophage collagenase has been characterized as a typical metal proteinase which catalyzes the cleavage of the native collagen molecule into three and one-quarter fragments. The extracellular accumulation and low activity in cell lysates suggest that collagenase is a secretion product of the stimulated macrophage. Prolonged secretion of the enzyme at a constant rate for more than 7 days in culture and its inhibition by cycloheximide provide evidence for biosynthesis in vitro. In contrast, secretion of collagenase is barely detectable from unstimulated macrophages which can, however, be stimulated to secret the enzyme by ingestion and intralysosomal storage of latex particles or dextran sulfate. Macrophages laden with latex, an undigestable particle, continue to release collagenase for at least 20 days. Several established mouse cell lines have also been examined for their capacity to secrete collagenase. Collagenase is one of a class of inducible neutral proteinases by which the activated macrophage can modify its extracellular environment.


Asunto(s)
Medios de Cultivo , Macrófagos/enzimología , Colagenasa Microbiana/metabolismo , Animales , Líquido Ascítico/citología , Células Cultivadas , Dextranos/farmacología , Electroforesis en Gel de Poliacrilamida , Femenino , Sangre Fetal/fisiología , Lactalbúmina/farmacología , Látex/administración & dosificación , Macrófagos/metabolismo , Ratones , Colagenasa Microbiana/análisis , Microesferas , Fagocitosis , Pinocitosis/efectos de los fármacos , Tioglicolatos/farmacología
12.
J Exp Med ; 140(6): 1482-97, 1974 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-4372292

RESUMEN

Rabbit synovial fibroblasts in monolayer culture secrete a specific collagenase and a neutral endopeptidase into their serum-free culture medium. The rate of secretion of these two enzymes is increased after the ingestion and storage of latex particles within the vacuolar system of the cells. The increased rates of secretion of the neutral enzymes are stable for over 2 wk in the absence of a further phagocytic bout. In constrast there is little change in the extracellular levels of two lysosomal hydrolases, cathepsin D and beta-glucuronidase. The increase in the secretory rates for the two neutral enzymes is related to the number of latex particles ingested by the cells, and increases of up to 12-fold over the nonphagocytosing cultures were observed. A variety of other materials including mycostatin particles and dextran sulfate also induced increases in the secretion of collagenase. These results are discussed in relation to the turnover of connective tissue matrix macromolecules.


Asunto(s)
Endocitosis , Colagenasa Microbiana/metabolismo , Péptido Hidrolasas/metabolismo , Fagocitosis , Animales , Radioisótopos de Carbono , Catepsinas/metabolismo , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Glucuronidasa/metabolismo , Látex , Microesferas , Conejos , Tasa de Secreción , Membrana Sinovial/citología
13.
J Exp Med ; 135(1): 21-44, 1972 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-4550608

RESUMEN

Phagocytosis of cholesterol-containing particles resulted in the formation of an intralysosomal cholesterol compartment. Cholesterol was excreted out of the macrophage with a single exponential rate which depended on the concentration of acceptor lipoproteins in the medium. Exchange kinetics performed on cells which had ingested particulate cholesterol suggested that excretion occurred by the same mechanism as exchange. Cholesterol esters as particulate albumin coacervates were taken up by macrophages and hydrolyzed by a lysosomal cholesterol esterase with optimal activity at pH 4.0. Cholesteryl linoleate was hydrolyzed much more readily than cholesteryl palmitate. The amount of cholesterol esterase and its specific activity increased during the in vitro cultivation of macrophages. Intralysosomally, cholesteryl linoleate and palmitate were hydrolyzed to free cholesterol which was excreted from the macrophage and recovered in the medium. Since cholesteryl linoleate was hydrolyzed more rapidly than free cholesterol was excreted into the medium, free cholesterol accumulated intralysosomally. Cholesteryl palmitate was hydrolyzed more slowly, and the rate of hydrolysis was limiting for excretion of the free cholesterol from within the lysosome.


Asunto(s)
Colesterol/metabolismo , Macrófagos/metabolismo , Fagocitosis , Fosfatasa Ácida/metabolismo , Isótopos de Carbono , Eritrocitos/metabolismo , Esterasas/metabolismo , Ésteres/metabolismo , Humanos , Hidrólisis , Técnicas In Vitro , Cinética , Ácidos Linoleicos , Lipoproteínas/metabolismo , Lisosomas/metabolismo , Macrófagos/enzimología , Macrófagos/fisiología , Microscopía Electrónica , Microscopía de Contraste de Fase , Ácidos Palmíticos , Fosfolípidos/metabolismo , Tritio
14.
J Exp Med ; 134(6): 1545-69, 1971 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-5126640

RESUMEN

The cholesterol metabolism of homogeneous populations of mouse peritoneal macrophages was evaluated under in vitro conditions. Macrophages are rich in free cholesterol and maintain a constant cholesterol to protein ratio (12 microg cholesterol/mg protein). No detectable cholesterol ester was present within the cell. More than 95% of total cholesterol was membrane associated and the majority was present in subcellular fractions containing lysosomes and plasma membrane. Less than 0.1% of cell cholesterol was synthesized from acetate-1-(14)C. During in vitro cultivation, macrophages rapidly exchanged their membrane cholesterol with that of lipoproteins of calf serum. About 30% of the cell cholesterol was exchanged per hour in 20% serum medium, and exchange was nearly complete by 5 hr. Exchange proceeded in a rapid exponential phase followed by a slower phase. Calculations based on a two compartment model indicated that the rapidly exchanging cholesterol compartment represented 60-70% of the total cell cholesterol, and the slowly exchanging compartment accounted for 30-40%. The relationship between serum lipoprotein concentration and exchange rate exhibited first-order kinetics. The rate was determined by thermal energy, in keeping with a Q(10) of 2, and an activation energy of 12 kcal/mole. Exchange was independent of bulk transport of lipoproteins by pinocytosis and phagocytosis, and was not linked to energy metabolism. The alpha-lipoproteins were the major class of proteins of calf serum participating in exchange.


Asunto(s)
Colesterol/metabolismo , Macrófagos/metabolismo , Fosfatasa Ácida/análisis , Animales , Electroforesis de las Proteínas Sanguíneas , Células Cultivadas , Centrifugación , Colesterol/análisis , Cromatografía de Gases , Cromatografía en Capa Delgada , Ésteres/análisis , Técnicas In Vitro , Cinética , Lipoproteínas/sangre , Macrófagos/citología , Ratones , Modelos Biológicos , Fracciones Subcelulares/metabolismo , Temperatura , Tritio
15.
J Exp Med ; 134(6): 1570-90, 1971 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-4942408

RESUMEN

Macrophage membrane cholesterol is present in two subcellular cholesterol pools, a rapidly exchanging compartment comprising about two-thirds of the total cholesterol, and a slowly exchanging compartment comprising one-third of the total. The morphological identification of the kinetically distinguishable pools proceeded by alteration of each compartment. Trypsin treatment markedly decreased the rate of cholesterol exchange without removing cholesterol from the membrane. Recovery of normal exchange rates took more than 7 hr and required protein synthesis. This suggested that a plasma membrane receptor is involved in positioning of lipoproteins for exchange, and is consistent with the plasma membrane localization of the rapidly exchanging compartment. Extensive pinocytosis by nondegradable dextran, dextran sulfate, or sucrose resulted in the accumulation of many secondary lysosomes, thus increasing the relative proportion of intracellular membranes. The measurable granule membrane area, cholesterol content, phospholipid content, and the relative size of the slowly exchanging cholesterol compartment all increased. The amount of intracellular membrane altered by extensive phagocytosis of latex particles also increased the size of the slowly exchanging cholesterol compartment. This suggested that the slowly exchanging pool of cholesterol represented the intracellular membranes primarily of lysosomal origin. Rabbit alveolar macrophages and thioglycollate-stimulated peritoneal macrophages contain many secondary lysosomes as a result of multiple bouts of in vivo phagocytosis and pinocytosis. In both of these cells the fast and slow pools are equal in size. The increased cholesterol content was attributable to the increase in the relative size of the slowly exchanging compartment. L-cells and melanoma cells also exchange their cholesterol with that of serum lipoproteins. Both cells contain few cholesterol-rich intracellular membranes, and had lower cellular cholesterol contents. In these cells the slowly exchanging pool was a minor contribution to cell cholesterol. Studies with these cells provided further evidence for the lysosomal membrane and plasma membrane localization of the slowly and rapidly exchanging cholesterol compartments.


Asunto(s)
Colesterol/metabolismo , Macrófagos/metabolismo , Fracciones Subcelulares/metabolismo , Animales , Dextranos/metabolismo , Técnicas In Vitro , Cinética , Células L/metabolismo , Látex , Lisosomas/metabolismo , Macrófagos/citología , Melanoma/metabolismo , Ratones , Microscopía Electrónica , Microscopía de Contraste de Fase , Microesferas , Mycobacterium bovis , Cavidad Peritoneal/citología , Fagocitosis , Pinocitosis , Alveolos Pulmonares/citología , Tioglicolatos/farmacología , Tritio , Tripsina/farmacología
16.
J Exp Med ; 142(2): 361-77, 1975 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-167096

RESUMEN

Thioglycolate-stimulated mouse peritoneal macrophages secrete a Proteinase which degrades insoluble elastin. There is little elastase activity in cell lysates but the bulk of the enzyme accumulates extracellularly during culture in serum-free medium. The secretion of elastase is sustained for over 12 days in culture and continued secretion of elastase requires protein synthesis. Unstimulated macrophages secrete very little elastase activity but can be triggered to secrete higher levels of this enzyme by phagocytosis and intracellular storage of latex particles. The macrophages elastase is a distinctive proteinase differing from the elastases of pancreas and granulocytes and is distinct from the other secreted proteinases of macrophages, namely, collagenase and plasminogen activator. The macrophages elastase is a serine proteinase and is inhibited by di-isopropyl phosphoro-fluoridate, ovoinhibitor, EDTA, dithiothretiol, and serum. Its activity is little affected by soybean trypsin inhibitor, turkey ovomucoid and chloromethyl ketones derived from tosyl lysine, tosyl phenylalanine, and acetyltetra alanine. Hydrolysis by macrophage elastase of chromogenic ester substrates for pancreatic elastase could not be detected. Elastase secretion by stimulated macrophages exceeds that by primary and established fibroblast cell strains. It is likely that elastase secretion by macrophages plays a major role in the pathogenesis of chronic destructive pulmonary diseases such as emphysema.


Asunto(s)
Macrófagos/enzimología , Elastasa Pancreática/metabolismo , Animales , Líquido Ascítico/citología , Células Cultivadas , Pruebas Enzimáticas Clínicas , Elastina , Esterasas/análisis , Femenino , Inmunodifusión , Látex , Ratones , Colagenasa Microbiana/análisis , Microesferas , Elastasa Pancreática/análisis , Fagocitosis , Cloruro de Sodio/farmacología , Dodecil Sulfato de Sodio/farmacología , Tioglicolatos/farmacología
17.
J Exp Med ; 152(6): 1527-36, 1980 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-6450258

RESUMEN

Thioglycollate-elicited mouse peritoneal macrophages were cultured in contact with the mixture of extracellular matrix proteins produced by rat smooth muscle cells in culture. Both live macrophages and their conditioned media hydrolyzed glycoproteins, elastin, and collagen. Live macrophages also degraded extracellular connective tissue proteins secreted by endothelial cells and fibroblasts. The glycoproteins in the matrix markedly inhibited the rate of digestion of the other macromolecules, particularly elastin. When plasminogen was added to the matrix, activation of plasminogen to plasmin resulted in the hydrolysis of the glycoprotein components, which then allowed the macrophage elastase easier access to its substrate, elastin. Thus, although plasmin has no direct elastinolytic activity, its presence accelerated the rate of hydrolysis of elastin and therefore the rate of matrix degradation. These findings may be important in an understanding of disease states, such as emphysema and atherosclerosis, that are characterized by the destruction of connective tissue.


Asunto(s)
Colágeno/metabolismo , Tejido Conectivo/metabolismo , Elastina/metabolismo , Glicoproteínas/metabolismo , Macrófagos/fisiología , Animales , Células Cultivadas , Espacio Extracelular/metabolismo , Fibrinolisina/metabolismo , Ratones , Músculo Liso/citología , Plasminógeno/metabolismo , Ratas , Tripsina/metabolismo
18.
J Exp Med ; 147(6): 1684-94, 1978 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-681878

RESUMEN

Glucocorticoid binding was measured in resident and thioglycollate-elicited mouse peritoneal macrophages, rabbit alveolar macrophages, and human monocytes. Two assays of binding were used--an assay with intact cells in suspension or monolayers, and an assay of cytosol and nuclear forms of glucocorticoid receptors. The mononuclear phagocytes contained approximately equal to 4--10 X 10(3) high affinity receptor sites per cell, with dissociation constants of approximately equal to 2--8 nM dexamethasone. The binding to the saturable sites was specific for steroids with glucocorticoid or antiglucocorticoid activity. Cortisol, corticosterone, and progesterone competed with dexamethasone for binding, whereas estradiol, dihydrotestosterone, and 11-epicortisol competed very little. Binding of dexamethasone to cytosol and nuclear forms of the receptor complex and temperature-sensitive translocation of cytosol forms to nuclear forms were shown. At 37 degrees C the predominant form of the hormone-receptor complex was nuclear. These results demonstrate that corticosteroids interact with macrophages at physiological concentrations.


Asunto(s)
Macrófagos/metabolismo , Monocitos/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Esteroides/metabolismo , Animales , Líquido Ascítico/citología , Núcleo Celular/metabolismo , Cortisona/metabolismo , Citoplasma/metabolismo , Dexametasona/metabolismo , Humanos , Ratones , Alveolos Pulmonares/citología , Conejos , Temperatura , Tioglicolatos/inmunología
19.
J Exp Med ; 152(6): 1537-53, 1980 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-7005386

RESUMEN

We have shown that macrophages in culture degrade the glycoproteins and amorphous elastin of insoluble extracellular matrices. Ultrastructural observation of the macrophage-matrix interaction revealed that connective tissue macromolecules were solubilized from the matrix extracellularly. At least part of the matrix breakdown was localized to the immediate vicinity of the cells, as shown by morphological and biochemical studies, although the rate of degradation correlated closely with the secretion of proteinases by various inflammatory stimuli in vivo, by glucocorticoids, prostaglandin E2 or colchicine, or by phagocytosis of latex, zymosan, or cholesterol-albumin complexes in culture was reflected in altered rates of glycoprotein and elastin degradation by the macrophages. Alteration of endocytosis and lysosomal digestion by cytochalasin B, NH4Cl, and proteinase inhibitors did not decrease the overall rate of matrix solubilization, but reduced the processing of the matrix fragments to peptides. Therefore, extracellular, pericellular, and lysosomal events each contribute to degradation of extracellular matrix macromolecules by inflammatory macrophages.


Asunto(s)
Tejido Conectivo/metabolismo , Espacio Extracelular/metabolismo , Macrófagos/fisiología , Endocitosis , Hidrólisis , Lisosomas/metabolismo , Macrófagos/ultraestructura , Microscopía Electrónica , Péptido Hidrolasas/metabolismo , Fagocitosis , Inhibidores de Proteasas/farmacología
20.
J Exp Med ; 152(5): 1340-57, 1980 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-7000966

RESUMEN

We have investigated the ability of neutral and lysosomal enzymes of mouse macrophages to degrade the insoluble extracellular matrices secreted by smooth muscle cells, endothelial cells, and fibroblasts. Matrices produced by smooth muscle cells contained glycoproteins, elastin, and collagens, but matrices of endothelial cells and fibroblasts contained no elastin. Sequential enzyme digestion of residual matrix revealed that plasmin, a product of macrophage plasminogen activation, degraded 50-70% of the glycoprotein in the matrices but did not degrade the elastin or the collagens. Purified macrophage elastase degraded glycoprotein and elastin components but had no effect on the collagens. The rate of elastin degradation by macrophage elastase was decreased in the presence of the glycoproteins. In contrast, human granulocyte elastase effectively degraded the matrix glycoproteins, elastin, and, to a lesser extent, collagens, Mammalian collagenase degraded only collagens. Conditioned medium from resident and inflammatory macrophages, containing mixtures of the secreted proteinases, degraded the glycoprotein and elastin components of the matrices. However, conditioned medium was less effective in degrading matrix than comparable amounts of purified macrophage elastase because > 90% of the elastase in the medium was in a latent form. Inclusion of plasminogen in the assays accelerated degradation. In the presence of plasminogen, glycoproteins were degraded readily by medium from P388D1, pyran copolymer-, thioglycollate-, and periodate-elicited macrophages and, to a lesser extent, by medium from endotoxin-elicited and resident macrophages; medium from P388D1, thioglycollate-, and periodate-elicited macrophages was most effective in elastin degradation, and resident, endotoxin-elicited and pyran copolymer-elicited macrophages degraded almost no elastin. The macrophage cathepsins D and B degraded all the matrix components at an optimum pH of 5.5 and acted with the secreted neutral proteinases to degrade the connective tissue macromolecules to amino acids and oligopeptides. These data indicate that macrophages at inflammatory sites contain and secrete proteolytic enzymes that could degrade the extracellular matrix.


Asunto(s)
Colágeno/metabolismo , Elastina/metabolismo , Endopeptidasas/metabolismo , Glicoproteínas/metabolismo , Macrófagos/enzimología , Animales , Concentración de Iones de Hidrógeno , Lisosomas/enzimología , Ratones , Elastasa Pancreática/metabolismo
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