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Enfermedad Granulomatosa Crónica/complicaciones , Enfermedad Granulomatosa Crónica/cirugía , Escisión del Ganglio Linfático , Linfadenitis/etiología , Linfadenitis/cirugía , Disección del Cuello , Adolescente , Adulto , Niño , Femenino , Humanos , Masculino , Cuello , Estudios Retrospectivos , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: The efficacy of granulocyte transfusions in patients with HLA alloimmunization is uncertain. A flow cytometric assay using dihydrorhodamine 123 (DHR), a marker for cellular NADPH oxidase activity, was used to monitor the differential survival of transfused oxidase-positive granulocytes in alloimmunized patients with chronic granulomatous disease (CGD). STUDY DESIGN AND METHODS: Ten patients with CGD and serious infections were treated with daily granulocyte transfusions derived from steroid and granulocyte-colony-stimulating factor-stimulated donors. The proportion of neutrophils with intact oxidase activity was quantitated by DHR fluorescence on samples drawn before and 1 hour after transfusion. The incidence of acute transfusion reactions was correlated with the results of DHR fluorescence and biweekly HLA serologic screening assays. RESULTS: Eight of 10 patients experienced acute adverse reactions in association with granulocyte transfusions. Four had only chills and/or fever, and four experienced respiratory compromise; all eight exhibited HLA alloimmunization. Mean (± SD) oxidase-positive cell recovery was 19.7 ± 17.4% (n = 15 transfusions) versus 0.95 ± 1.59% (n = 16) in the absence and presence of HLA allosensitization, respectively (p < 0.01). Greater than 1% in vivo recovery of DHR-enhancing donor granulocytes was strongly correlated with lack of HLA alloimmunization. CONCLUSION: The ability to detect DHR-positive donor granulocytes by flow cytometry is strongly correlated with absence of HLA alloimmunization and lack of acute reactions to granulocyte transfusions in patients with CGD. If HLA antibodies are present and the survival of donor granulocytes is low by DHR analysis, transfusions should be discontinued, avoiding a therapy associated with high risk and unclear benefit.
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Granulocitos/trasplante , Enfermedad Granulomatosa Crónica/terapia , Transfusión de Leucocitos/métodos , Adolescente , Niño , Preescolar , Femenino , Citometría de Flujo , Humanos , Masculino , Neutrófilos/citología , Adulto JovenRESUMEN
Chronic granulomatous disease (CGD) is caused by a congenital defect in phagocyte reduced nicotinamide dinucleotide phosphate (NADPH) oxidase production of superoxide and related species. It is characterized by recurrent life-threatening bacterial and fungal infections and tissue granuloma formation. We have created a mouse model of CGD by targeted disruption of p47phox, one of the genes in which mutations cause human CGD. Identical to the case in human CGD, leukocytes from p47phox-/- mice produced no superoxide and killed staphylococci ineffectively. p47phox-/- mice developed lethal infections and granulomatous inflammation similar to those encountered in human CGD patients. This model mirrors human CGD and confirms a critical role for the phagocyte NADPH oxidase in mammalian host defense.
Asunto(s)
Enfermedad Granulomatosa Crónica/genética , NADH NADPH Oxidorreductasas/deficiencia , NADPH Deshidrogenasa/fisiología , Fagocitos/enzimología , Fosfoproteínas/fisiología , Absceso/etiología , Animales , Infecciones Bacterianas/inmunología , Secuencia de Bases , Susceptibilidad a Enfermedades , Activación Enzimática , Femenino , Granuloma/etiología , Enfermedad Granulomatosa Crónica/inmunología , Enfermedad Granulomatosa Crónica/patología , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Micosis/inmunología , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/metabolismo , NADPH Deshidrogenasa/deficiencia , NADPH Deshidrogenasa/genética , NADPH Oxidasas , Fagocitosis , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Especies Reactivas de Oxígeno/metabolismo , Estallido Respiratorio , Superóxidos/metabolismoRESUMEN
Histamine diphosphate was shown to selectively attract human eosinophils from mixed granulocyte populations when over 20% eosinophils were used in a modified Boyden chamber chemotactic assay system. This effect of histamine is abolished by incubation with diamine oxidase (histaminase) and was generated by decarboxylation of L-histidine. A linear dose dependent increase in eosinophil migration was observed between 3 X 10(-7) M and 1.25 X 10(-6) M, while higher concentrations of histamine inhibited the migration of eosinophils. The attractant activity of histamine was not inhibited by H-1 or H-2 receptor antagonists, however, the inhibition of migration observed at higher histamine concentrations was reversed by metiamine, an H-2 receptor antagonist. The effects of histamine upon eosinophil migration were demonstrable using three different assays: (a) counting cells that had traversed 5-mum pore, 12-mum thick polycarbonate filters, (b) counting cells that had migrated various distances into a 3-mum pore, 145-mum cellulose nitrate filters, or (c) measuring the number of cells that had traversed an upper polycarbonate filter and migrated into a lower cellulose nitrate filter using 15Cr-labeled cells. The ability of histamine to enhance eosinophil migration was shown to be dependent upon the presence of a concentration gradient; histamine did not cause a dose-dependent increase in random motility. Furthermore, preincubation of the eosinophils with histamine deactivate the cells to further stimulation by histamine or by C5a. It is concluded that in low doses histamine is a chemoattractant for human eosinophils, while in higher doses histamine inhibits eosinophil migration. These observations may relate to the influx and localization of eosinophils in immediate hypersensitivity reactions.
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Quimiotaxis/efectos de los fármacos , Eosinófilos/efectos de los fármacos , Histamina/farmacología , Amina Oxidasa (conteniendo Cobre) , Relación Dosis-Respuesta a Droga , Antagonistas de los Receptores Histamínicos H1/farmacología , Histidina/metabolismo , Humanos , Metiamida/farmacología , Pirilamina/farmacologíaRESUMEN
Neutrophil-specific granule deficiency (SGD) is a rare disorder characterized by recurrent pyogenic infections, defective neutrophil chemotaxis and bactericidal activity, and lack of neutrophil secondary granule proteins. CCAAT/enhancer binding protein (C/EBP)epsilon, a member of the leucine zipper family of transcription factors, is expressed primarily in myeloid cells, and its knockout mouse model possesses distinctive defects, including a lack of neutrophil secondary granule proteins. Sequence analysis of the genomic DNA of a patient with SGD revealed a five-basepair deletion in the second exon of the C/EBPepsilon locus. The predicted frame shift results in a truncation of the 32-kD major C/EBPepsilon isoform, with loss of the dimerization domain, DNA binding region, and transcriptional activity. The multiple functional defects observed in these early neutrophil progenitor cells, a consequence of C/EBPepsilon deficiency, define SGD as a defect in myelopoiesis and establish the requirement for C/EBPepsilon for the promyelocyte-myelocyte transition in myeloid differentiation.
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Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Síndromes de Inmunodeficiencia/genética , Mutación , Neutrófilos/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Animales , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT , Gránulos Citoplasmáticos/patología , Gránulos Citoplasmáticos/fisiología , Cartilla de ADN/genética , Células HeLa , Humanos , Síndromes de Inmunodeficiencia/patología , Síndromes de Inmunodeficiencia/fisiopatología , Ratones , Ratones Noqueados , Neutrófilos/patología , Proteínas Nucleares/deficiencia , Reacción en Cadena de la PolimerasaRESUMEN
In the present study, we demonstrated that expression of the LFA-1 molecule is necessary for cell fusion and syncytia formation in human immunodeficiency virus (HIV)-infected CD4+ T lymphocytes. In contrast, the lack of expression of LFA-1 does not influence significantly cell-to-cell transmission of HIV. In fact, LFA-1- T lymphocytes obtained from a leukocyte adhesion deficiency patient were unable to fuse and form syncytia when infected with HIV-1 or HIV-2, despite the fact that efficiency of HIV infection (i.e., virus entry, HIV spreading, and levels of virus replication) was comparable with that observed in LFA-1+ T lymphocytes. In addition, we provide evidence that LFA-1 by mediating cell fusion contributes to the depletion of HIV-infected CD4+ T lymphocytes in vitro.
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Linfocitos T CD4-Positivos/microbiología , VIH-1/fisiología , VIH-2/fisiología , Antígeno-1 Asociado a Función de Linfocito/fisiología , Anticuerpos Monoclonales , Fusión Celular , Células Cultivadas , Genes Virales/genética , Células Gigantes , VIH-1/genética , VIH-2/genética , Humanos , Activación de Linfocitos , Antígeno-1 Asociado a Función de Linfocito/deficiencia , Antígeno-1 Asociado a Función de Linfocito/genética , Fitohemaglutininas , Reacción en Cadena de la Polimerasa , Replicación ViralRESUMEN
Considerable evidence suggests that Ca2+ modulates endothelial cell metabolic and morphologic responses to mediators of inflammation. We have used the fluorescent Ca2+ indicator, quin2, to monitor endothelial cell cytosolic free Ca2+, [Ca2+]i, in cultured human umbilical vein endothelial cells. Histamine stimulated an increase in [Ca2+]i from a resting level of 111 +/- 4 nM (mean +/- SEM, n = 10) to micromolar levels; maximal and half-maximal responses were elicited by 10(-4) M and 5 X 10(-6) M histamine, respectively. The rise in [Ca2+]i occurred with no detectable latency, attained peak values 15-30 s after addition of stimulus, and decayed to a sustained elevation of [Ca2+]i two- to threefold resting. H1 receptor specificity was demonstrated for the histamine-stimulated changes in [Ca2+]i. Experiments in Ca2+-free medium and in the presence of pyrilamine or the Ca2+ entry blockers Co2+ or Mn2+, indicated that Ca2+ mobilization from intracellular pools accounts for the initial rise, whereas influx of extracellular Ca2+ and continued H1 receptor occupancy are required for sustained elevation of [Ca2+]i. Ionomycin-sensitive intracellular Ca2+ stores were completely depleted by 4 min of exposure to 5 X 10(-6) M histamine. Verapamil or depolarization of endothelial cells in 120 mM K+ did not alter resting or histamine-stimulated [Ca2+]i, suggesting that histamine-elicited changes are not mediated by Ca2+ influx through voltage-gated channels. Endothelial cells grown on polycarbonate filters restricted the diffusion of a trypan blue-albumin complex; histamine (through an H1-selective effect) promoted trypan blue-albumin diffusion with a concentration dependency similar to that for the histamine-elicited rise in [Ca2+]i. Exposure of endothelial cells to histamine (10(-5) M) or ionomycin (10(-7) M) was associated with a decline in endothelial F-actin (relative F-actin content, 0.76 +/- 0.07 vs. 1.00 +/- 0.05; histamine vs. control, P less than 0.05; relative F-actin content, 0.72 +/- 0.06 vs. 1.00 +/- 0.05; ionomycin vs. control, P less than 0.01). The data support a role for cytosolic calcium in the regulation of endothelial shape change and vessel wall permeability in response to histamine.
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Actinas/metabolismo , Calcio/metabolismo , Endotelio/metabolismo , Histamina/farmacología , Receptores Histamínicos H1/fisiología , Receptores Histamínicos/fisiología , Albúmina Sérica Bovina/metabolismo , Aminoquinolinas , Células Cultivadas , Citosol/metabolismo , Difusión , Endotelio/efectos de los fármacos , Humanos , Cinética , Venas UmbilicalesRESUMEN
We have recently reported a specific dose-dependent stimulation of posttranslational incorporation of tyrosine into tubulin alpha-chains of rabbit peritoneal leukocytes as induced by the synthetic peptide chemoattractant formyl-methionyl-leucyl-phenylalanine (FMLP). The present study reports a similar, specific stimulation of tubulin tyrosinolation in human polymorphonuclear leukocytes (PMN). When compared to normal PMN, both the resting and FMLP-stimulated levels of posttranslational tyrosine incorporation were two- to threefold higher in PMN of three patients with the Chediak-Higashi syndrome (CHS). The concentration of cellular tubulin and the specific activity of tubulin tyrosine ligase were similar in PMN of CHS patients and normal donors and resembled that of other non-neuronal cells. The high levels of tyrosine incorporation in PMN of CHS patients were normalized by the administration of ascorbate, both in vitro and in in vivo experiments. In vitro addition of ascorbate also inhibited the FMLP-induced stimulation of tyrosine incorporation in both normal and CHS cells. Normalization of higher levels of tyrosine incorporation in PMN of CHS patients and the inhibition of FMLP-induced stimulation of tubulin tyrosinolation in normal and CHS cells as observed with ascorbate could also be affected by other reducing agents such as reduced glutathione, cysteine, or dithiothreitol. These results suggest a possible relationship between cellular redox and tubulin tyrosinolation in PMN.
Asunto(s)
Síndrome de Chediak-Higashi/sangre , Metionina/análogos & derivados , N-Formilmetionina/análogos & derivados , Neutrófilos/metabolismo , Oligopéptidos/farmacología , Tubulina (Proteína)/metabolismo , Tirosina/metabolismo , Ácido Ascórbico/farmacología , Humanos , N-Formilmetionina/farmacología , N-Formilmetionina Leucil-Fenilalanina , Oxidación-Reducción , Péptido Hidrolasas/sangre , Procesamiento Proteico-Postraduccional , Tubulina (Proteína)/sangreRESUMEN
Potassium pyroantimonate was used to localize sites of bound cations in human neutrophils under conditions of random migration, stimulated random migration (chemokinesis), and directed migration (chemotaxis). The cells were placed in a standard chamber in which 0.45-micron micropore filters separated the cells from the stimulus (buffer, Escherichia coli endotoxin-activated serum or the synthetic chemotactic peptide N-formyl-Met-Leu-Phe). The small pore filters permitted pseudopod formation but impeded cell imgration through the filter. Cells examined under all conditions had electron-dense precipitates of antimonate salts in some granules. However, antimonate deposits were localized in the condensed chromatin of the nucleus during random migration and associated to a large extent with the uncondensed nuclear chromatin during chemokinesis and chemotaxis. Under conditions of chemokinesis deposition of antimonate procipitates appeared on the cytoplasmic side of the plasma membrane of neutrophils whereas under conditions of chemotaxis cation deposits beneath the cell membrane were localized to the pseudopods which were directed toward the chemoattractant. In addition to endotoxin-activated serum, concentrations of N-formyl-Met-Leu-Phe which caused neutrophil chemotaxis (10(-8) M) also caused cation deposition beneath the cell membrane at the leading end of the cell regardless of whether albumin was present in the incubation media. However, with higher concentrations of the synthetic peptide (10(-5) M) which caused granule release and were not chemotactic, submembranous cation deposition was not seen. EDTA (10 mM) and EGTA (10 mM) removed nuclear, granular, and submembranous cation deposits from neutrophils examined under conditions of chemotaxis. X-ray microprobe analysis of antimonate deposits revealed the possible presence of calcium but did not detect sodium or magnesium. The data indicate that chemotactic factors induce submembranous deposition of cations, most likely Ca++, which localize to the leading edge of cells exposed to a gradient of chemoattractant.
Asunto(s)
Calcio/aislamiento & purificación , Quimiotaxis de Leucocito , Neutrófilos/fisiología , Antimonio/farmacología , Núcleo Celular/análisis , Quimiotaxis de Leucocito/efectos de los fármacos , Gránulos Citoplasmáticos/análisis , Humanos , Neutrófilos/efectos de los fármacos , Neutrófilos/ultraestructura , Péptidos/farmacología , Seudópodos/análisisRESUMEN
Optimal human granulocyte chemotaxis has been shown to require both calcium and magnesium. Exposure of granulocytes to three different chemotactic factors (C5a, kallikrein, and dialyzable transfer factor) yielded a rapid calcium release, depressed calcium uptake, and was associated with a shift of calcium out of the cytoplasm and into a granule fraction. Colchicine, sodium azide, and cytochalasin B, in concentrations that inhibited chemotaxis, also inhibited calcium release while low concentrations of cytochalasin B, which enhanced chemotaxis, also enhanced calcium release. Microtubule assembly was visualized both in cells suspended in C5a without a chemotactic gradient and in cells actively migrating through a Micropore filter. The data suggest microtubule assembly is regulated, at least, in part, by the level of cytoplasmic calcium. It is proposed that asymmetric assembly of microtubules may be instrumental in imparting the net vector of motion during chemotaxis.
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Calcio/metabolismo , Quimiotaxis , Leucocitos/citología , Magnesio/metabolismo , Microtúbulos , Azidas/farmacología , Radioisótopos de Calcio , Cromatografía , Colchicina/farmacología , Proteínas del Sistema Complemento/farmacología , Citocalasina B/farmacología , Citoplasma/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Inmunidad Materno-Adquirida , Calicreínas/farmacología , Cinética , Filtros Microporos , TemperaturaRESUMEN
Serotonin, ascorbic acid, and carbamylcholine enhanced the chemotactic responsiveness of human monocytes to endotoxin-treated serum. These agents caused significant accumulation of cyclic GMP in monocytes. PMN leukocyte chemotaxis was also enhanced by these agents although significant increases in cyclic GMP were not demonstrated.
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Ácido Ascórbico/farmacología , Carbacol/farmacología , Quimiotaxis/efectos de los fármacos , GMP Cíclico/metabolismo , Leucocitos/efectos de los fármacos , Serotonina/farmacología , Bradiquinina/farmacología , Histamina/farmacología , Humanos , Leucocitos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Neutrófilos/efectos de los fármacosRESUMEN
The electrophysiology of chemotactic factor interaction with cultured human macrophages was investigated with standard intracellular recording techniques. In initial studies, E. coli endotoxin-activated serum, added to cell cultures during intracellular recordings, caused membrane hyperpolarizations which were greater than 30 s in duration, 10-50 mV in amplitude, and associated with decreased membrane resistance. Control serum produced smaller hyperpolarizations lasting 10-20 s and 5-30 m V in amplitude. Endotoxin-activated human serum deficient in the third complement component (C3) did not produce hyperpolarizations unless the serum was reconstituted with C3 before activation. Fractionation of normal activated serum by molecular seive chromatography (G-75 Sephadex) indicated that only fractions that eluted with an estimated molecular weight of 12,500 produced membrane potential changes. The active material that was chemotactic for the macrophages was identified as the small molecular weight cleavage product of C5, C5a, by heat stability (30 min at 56 degrees C) and inactivation by goat antisera to human C5 but not C3. 17 percent of macrophages stimulated with C5a exhibited a biphasic response characterized by a small (2-6 mV), brief (1-10 s) depolarization associated with a decreased membrane resistance preceding the larger and prolonged hyperpolarizations. Magnesium-ethylene glycol bis[beta-aminoethyl ether]N,N'-tetraacetic acid (Mg [2.5 mM]-EGTA [5.0 mM]) blocked the C5a-evoked potential changes, whereas colchine (10(- 6)M) and cytochalasin B (3.0 mug/ml did not. Hydrocortisone sodium succinate (0.5 mg/ml) decreased the percentage of cells responding to C5a. In related studies, synthetic N-formyl methionyl peptide (f-met-leu-phe), which had chemotactic activity for cultured macrophages, produced similar membrane potential changes. Repeated exposure of macrophages to C5a or f- met-leu-phe resulted in desensitization to the same stimulus. Simultaneous photomicroscope and intracellular recording studies during macrophage stimulation with chemotactic factor demonstrated that the membrane potential changes preceded membrane spreading, ruffling, and pseudopod formation. These observations demonstrate that ion fluxes associated with membrane potential changes are early events in macrophage activation by chemotactic factors
Asunto(s)
Quimiotaxis , Complemento C5 , Macrófagos/fisiología , Potenciales de la Membrana/efectos de los fármacos , Péptidos/farmacología , Colchicina/farmacología , Complemento C3 , Citocalasina B/farmacología , Ácido Egtácico/farmacología , Endotoxinas/farmacología , Humanos , Hidrocortisona/farmacología , Macrófagos/efectos de los fármacosRESUMEN
Orientation of nucleus, centriole, microtubules, and microfilaments within human neutrophils in a gradient of chemoattractant (5 percent Escherichia coli endotoxin-activated serum) was evaluated by electron microscopy. Purified neutropils (hypaque-Ficoll) were placed in the upper compartment of chemotactic chambers. Use of small pore (0.45 mum) micropore filters permitted pseudopod penetration, but impeded migration. Under conditions of chemotaxis with activated serum beneath the filter, the neutrophil population oriented at the filter surface with nuclei located away from the stimulus, centrioles and associated radial array of microtubules beneath the nuclei, and microfilament-rich pseudopods penetrating the filter pores. Reversal of the direction of the gradient of the stimulus (activated serum above cells) resulted in a reorientation of internal structure which preceded pseudopod formation toward the activated serum and migration off the filter. Coordinated orientation of the entire neutrophil population did not occur in buffer (random migration) or in a uniform concentration of activated serum (activated random migration). Conditions of activated random migration resulted in increased numbers of cells with locomotory morphology, i.e. cellular asymmetry with linear alignment of nucleus, centriole, microtubule array, and pseudopods. Thus, activated serum increased the number of neutrophils exhibiting locomotory morphology, and a gradient of activated serum induced the alignment of neutrophils such that this locomotory morphology was uniform in the observed neutrophil populayion. In related studies, cytochalasin B and colchicines were used to explore the role of microfilaments and microtubules in the neutrophil orientation and migration response to activated serum. Cytochalasin B (3.0 mug/ml) prevented migration and decreased the microfilaments seen, but allowed normal orientation of neutrophil structures. In an activated serum gradient, colchicines, but not lumicolchicine, decreased the orientation of nuclei and centrioles, and caused a decrease in centriole-associated microtubules in concentrations as low as 10(-8) to 10(-7) M. These colchicines effects were associated with the rounding of cells and impairment of pseudopod formation. The impaired pseudopod formation was characterized by an inability to form pseudopods in the absence of a solid substrate, a formation of narrow pseudopods within a substrate, and a defect in pseudopod orientation in an activated serum gradient. Functional studies of migration showed that colchicines, but not lumicolchicine, minimally decreased activated random migration and markedly inhibited directed migration, but had not effect on random migration. These studies show that, although functioning microfilaments are probably necessary for neutrophil migration, intact microtubules are essential for normal pseudopod formation and orientation, and maximal unidirectional migration during chemotaxis.
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Quimiotaxis , Citoplasma/fisiología , Citoesqueleto/fisiología , Microtúbulos/fisiología , Neutrófilos/fisiología , Sangre , Núcleo Celular , Quimiotaxis/efectos de los fármacos , Colchicina/farmacología , Citocalasina B/farmacología , Humanos , Neutrófilos/efectos de los fármacos , Neutrófilos/ultraestructura , Organoides/fisiología , SeudópodosRESUMEN
Chronic granulomatous diseases of childhood (CGD) are a group of disorders of phagocytic cell superoxide (O2.-) production (respiratory burst). Anion exchange chromatography separated from normal neutrophil cytosol a 47-kilodalton neutrophil cytosol factor, NCF-1, that restored activity to defective neutrophil cytosol from most patients with autosomally inherited CGD in a cell-free O2.--generating system. A 65-kilodalton factor, NCF-2, restored activity to defective neutrophil cytosol from one patient with autosomal CGD. NCF-1, NCF-2, and a third cytosol fraction, NCF-3, were inactive alone or in pairs, but together replaced unfractionated cytosol in cell-free O2.- generation. Neutrophils deficient in NCF-1, but not NCF-2, did not phosphorylate the 47-kilodalton protein. It is proposed that NCF-1, NCF-2, and NCF-3 are essential for generation of O2.- by phagocytic cells and that genetic abnormalities of these cytosol components can result in the CGD phenotype.
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Enfermedad Granulomatosa Crónica/metabolismo , Neutrófilos/metabolismo , Superóxidos/biosíntesis , Western Blotting , Membrana Celular/metabolismo , Citosol/metabolismo , Humanos , Técnicas In Vitro , Peso Molecular , Fosfoproteínas/metabolismoRESUMEN
A 47-kilodalton neutrophil cytosol factor (NCF-47k), required for activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase superoxide (O2-.) production, is absent in most patients with autosomal recessive chronic granulomatous disease (AR-CGD). NCF-47k cDNAs were cloned from an expression library. The largest clone predicted a 41.9-kD protein that contained an arginine and serine-rich COOH-terminal domain with potential protein kinase C phosphorylation sites. A 33-amino acid segment of NCF-47k shared 49% identity with ras p21 guanosine triphosphatase activating protein. Recombinant NCF-47k restored O2-. -producing activity to AR-CGD neutrophil cytosol in a cell-free assay. Production of active recombinant NCF-47k will enable functional regions of this molecule to be mapped.
Asunto(s)
ADN/genética , Enfermedad Granulomatosa Crónica/genética , NADH NADPH Oxidorreductasas/metabolismo , Neutrófilos/metabolismo , Fosfoproteínas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Clonación Molecular , Enfermedad Granulomatosa Crónica/enzimología , Humanos , Immunoblotting , Datos de Secuencia Molecular , NADPH Oxidasas , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Superóxidos/metabolismoRESUMEN
Chronic granulomatous diseases (CGDs) are characterized by recurrent infections resulting from impaired superoxide production by a phagocytic cell, nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) oxidase. Complementary DNAs were cloned that encode the 67-kilodalton (kD) cytosolic oxidase factor (p67), which is deficient in 5% of CGD patients. Recombinant p67 (r-p67) partially restored NADPH oxidase activity to p67-deficient neutrophil cytosol from these patients. The p67 cDNA encodes a 526-amino acid protein with acidic middle and carboxyl-terminal domains that are similar to a sequence motif found in the noncatalytic domain of src-related tyrosine kinases. This motif was recently noted in phospholipase C-gamma, nonerythroid alpha-spectrin (fodrin), p21ras-guanosine triphophatase-activating protein (GAP), myosin-1 isoforms, yeast proteins cdc-25 and fus-1, and the 47-kD phagocyte oxidase factor (p47), which suggests the possibility of common regulatory features.
Asunto(s)
NADH NADPH Oxidorreductasas/genética , Neutrófilos/enzimología , Proteínas Proto-Oncogénicas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Enfermedad Granulomatosa Crónica/sangre , Enfermedad Granulomatosa Crónica/enzimología , Enfermedad Granulomatosa Crónica/genética , Humanos , Datos de Secuencia Molecular , NADH NADPH Oxidorreductasas/sangre , NADPH Oxidasas , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas pp60(c-src) , Homología de Secuencia de Ácido NucleicoRESUMEN
Chemotactic factors decrease the negative surface charge of neutrophils (polymorphonuclear leukocytes [PMN]) and this has been speculated to be important in PMN margination and aggregation in vivo. PMN adherence and aggregation are also enhanced by degranulation of lysosomal enzymes. To further assess the possible relationship between degranulation, surface charge, adherence, and aggregation, human peripheral blood PMN (isolated by Hypaque-Ficoll and dextran sedimentation) were exposed to the secretagogues ionophore A23187, phorbol myristate acetate, concanavalin A, and chemotactic factors (partially purified C5a or the synthetic peptide f-met-leu-phe) plus cytochalasin B. Surface charge was measured in a cytopherometer. After incubation of PMN with secretagogues, PMN surface charge was decreased to a greater extent than incubation of PMN with chemotactic factors. The decreased surface charge induced by f-met-leu-phe plus cytochalasin B required both extracellular calcium and magnesium. The ionophore A23187-induced surface charge changes were dependent on extracellular calcium but not magnesium whereas the phorbol myristate acetate effect was only partially dependent on Ca(++) and Mg(++). The surface charge changes induced by secretagogues were related to both the amount of lysozyme released and to the increased adhesiveness of cells to plastic surfaces. These observations indicate exocytosis of lysosomal granule contents is associated with decreases in neutrophil surface charge, and there appears to be a correlation between decreases in surface charge and facilitation of neutrophil aggregation and adhesiveness. However, a causal relationship between these events has not been established, and the relationship may be simply temporal.
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Adhesión Celular , Lisosomas/metabolismo , Potenciales de la Membrana , Neutrófilos/fisiología , Calcimicina/farmacología , Calcio/farmacología , Adhesión Celular/efectos de los fármacos , Factores Quimiotácticos/farmacología , Concanavalina A/farmacología , Citocalasina B/farmacología , Humanos , Técnicas In Vitro , Magnesio/farmacología , Potenciales de la Membrana/efectos de los fármacos , Superóxidos/sangre , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The chemotactic responsiveness of the neutrophils of 10 patients with the hyperimmunoglobulin E-recurrent infection syndrome (HIE) were compared with neutrophils from normal volunteers over a 10-mo period. HIE neutrophils as a group displayed significantly less chemotactic motility than control neutrophils. The data from individual patients were variable, being normal or abnormal on different days. Mononuclear cells from HIE patients, when cultured for 24 h in the absence of serum or a mitogen, produced a factor that inhibited normal neutrophil and monocyte chemotaxis. Mononuclear cells from normal volunteers with and without atopy or from patients with parasites or bacterial infections did not produce such an inhibitory factor. The production of this chemotactic inhibitory factor in vitro was variable over time, but it correlated with the presence of an in vitro neutrophil chemotactic defect. The chemotactic inhibitory factor was partially purified and was found to contain protein, to be stable at 56 degrees C, and to have a molecular weight of approximately 61,000. Irreversible inhibitors of serine esterases do not inactivate the factor. The factor is produced by esterase-negative mononuclear cells and is not toxic to neutrophils. This chemotactic inhibitory factor may be the basis of the variable chemotactic defect in HIE neutrophils.
Asunto(s)
Hipergammaglobulinemia/inmunología , Inmunoglobulina E , Linfocinas/biosíntesis , Monocitos/inmunología , Adolescente , Adulto , Infecciones Bacterianas/inmunología , Quimiotaxis de Leucocito , Niño , Femenino , Humanos , Linfocinas/aislamiento & purificación , Masculino , Persona de Mediana Edad , Neutrófilos/inmunología , SíndromeRESUMEN
After circulating in the vascular system a short time, polymorphonuclear leukocytes (PMN) migrate to extravascular sites in response to chemotactic stimuli. Prestimulation of PMN in vitro by secretagogues has been shown to increase their number of N-formylmethionylleucylphenylalanine (fmet-leu-phe) and complement component C3bi (CR3) receptors. We investigated whether the same phenomenon occurred in vivo, comparing characteristics of human skin chamber and guinea pig peritoneal exudate and blood PMN. Exudate PMN of both species contained approximately 28% less of the specific granule marker vitamin B12-binding protein (P less than 0.01) but a similar amount of the azurophil granule marker beta-glucuronidase. The total number of fmet-leu-phe receptors was 5.9 times higher in guinea pig exudate than in blood PMN (P less than 0.01) and 2.9 times higher in human exudate than in blood PMN (P less than 0.02). All exudate PMN and most blood PMN preparations showed a high affinity receptor (Kd approximately 2.3 X 10(-8) M) and a low affinity receptor (approximately 1.5 X 10(-7) M). The upregulation of fmet-leu-phe receptors in exudate PMN correlated with an improved responsiveness to fmet-leu-phe induced membrane depolarization, oxidative metabolism, and chemotaxis. In addition, the concentration of fmet-leu-phe that produced a half-maximal response of chemotaxis, superoxide production, and membrane potential depolarization was 10-fold lower in exudate PMN than in blood PMN. Human exudate PMN had a twofold increased C3bi receptor expression compared with blood PMN. Thus, a preferential loss of specific granules is associated with increased number of high and low affinity fmet-leu-phe receptors and increased C3bi receptor expression not only in vitro, but also in vivo. The data indicate that exudation primes PMN for their subsequent responsiveness to fmet-leu-phe, a modification that may be crucial for efficient antimicrobial host defense.
Asunto(s)
Quimiotaxis de Leucocito , Exudados y Transudados/inmunología , Neutrófilos/inmunología , Receptores de Complemento/inmunología , Animales , Gránulos Citoplasmáticos/ultraestructura , Cobayas , Humanos , Peróxido de Hidrógeno/metabolismo , Memoria Inmunológica , Potenciales de la Membrana , N-Formilmetionina Leucil-Fenilalanina/inmunología , Neutrófilos/fisiología , Neutrófilos/ultraestructura , Receptores de Formil Péptido , Receptores Inmunológicos/inmunología , Superóxidos/metabolismoRESUMEN
A specific stimulation of tubulin tyrosinolation in human polymorphonuclear leukocytes (PMN) is induced by the synthetic peptide chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe), and this stimulation of tyrosinolation in PMN is completely inhibited in the presence of various reducing agents. Further studies to characterize the mechanism of stimulation of tyrosinolation in PMN have revealed that conditions that inhibited the respiratory burst in stimulated PMN, e.g., an anaerobic atmosphere, or addition of antioxidants such as cysteamine, azide, or 2,3-dihydroxybenzoic acid, also inhibited the peptide-induced stimulation of tyrosinolation in these cells. Moreover, the sulfhydryl reagent, N-ethylmaleimide, depressed tyrosinolation in resting PMN and completely inhibited the fMet-Leu-Phe-induced stimulation. In contrast, addition of diamide, which preferentially oxidizes cellular glutathione, significantly stimulated tyrosinolation both in resting and fMet-Leu-Phe-stimulated PMN. Furthermore, resting levels of tyrosinolation in seven patients with chronic granulomatous disease (CGD), whose oxidative metabolism is severely depressed, were 35-45% lower (P less than 0.01). Most strikingly, PMN from CGD patients failed to respond to fMet-Leu-Phe or the Ca2+-ionophore A23187, which also induced stimulation of tyrosinolation in normal resting PMN. Methylene blue normalized the depressed tyrosinolation in resting CGD PMN, although it did not increase tyrosinolation in stimulated PMN. These results are consistent with the idea that the characteristic activation of the oxidative metabolism and the associated changes in the redox state in stimulated PMN are coupled to the induction of stimulation of tubulin tyrosinolation in these cells.