RESUMO
We have earlier shown through electron spin resonance (ESR) studies of leukocytes that membranes of cells from both Chediak-Higashi syndrome (CHS) mice and humans have abnormally high fluidity. We have extended our studied to erythrocytes. Erythrocytes were labeled with the nitroxide-substituted analogue of stearic acid, 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl, and ESR spectra were obtained. Order parameter, S, at 23 degrees C, was 0.661 and 0.653 for erythrocytes of normal and CHS mice (P less than 0.001). S was 0.684 for normal human erythrocytes and 0.675 (P less than 0.001) for CHS erythrocytes at 25 degrees C. Because S varies inversely to fluidity, these results indicate that CHS erythrocytes tend to have higher fluidity than normal. In vitro treatment of both mice and human CHS erythrocytes with 10 mM ascorbate returned their membrane fluidity to normal. We prepared erythrocyte ghosts and extracted them with CHCl3:CH3OH (2:1). Gas-liquid chromatography analysis showed a greater number of unsaturated fatty acids for CHS. The average number of double bonds detected in fatty acids for mice on a standard diet was 1.77 for normal and 2.02 for CHS (P less than 0.04); comparison of human erythrocytes from one normal control and one CHS patient showed a similar trend. Our results suggest that an increased proportion of unsaturated fatty acids may contribute to increased fluidity of CHS erythrocytes. Our observation that both leukocytes and erythrocytes of CHS have abnormal fluidity indicates that CHS pathophysiology may relate to a general membrane disorder.
Assuntos
Síndrome de Chediak-Higashi/sangue , Membrana Eritrocítica/fisiologia , Eritrócitos/fisiologia , Fluidez de Membrana , Lipídeos de Membrana/sangue , Animais , Proteínas Sanguíneas/análise , Colesterol/sangue , Membrana Eritrocítica/análise , Ácidos Graxos/sangue , Camundongos , Fosfolipídeos/sangueRESUMO
The intact leukocytes of two children with chronic granulomatous disease fail to reduce nitroblue tetrazolium during phagocytosis. This is due to defective operation of an oxidase of reduced nicotinamide adenine dinucleotide that is insensitive to cyanide and that indirectly stimulates the oxidation of glucose-6-phosphate in leukocytes. Such leukocytes undergo no increase in oxygen consumption or in activity of the hexose monophosphate shunt during phagocytosis, although lactate production is normal. The addition of nitroblue tetrazolium to a leukocyte suspension appears to provide a sensitive diagnostic screening test for this disease.
Assuntos
Granuloma/diagnóstico , Leucócitos/metabolismo , Oxirredutases/metabolismo , Fagocitose , Sais de Tetrazólio/metabolismo , Isótopos de Carbono , Criança , Doença Crônica , Hexosefosfatos/metabolismo , Humanos , Lactatos/biossíntese , Consumo de Oxigênio , Fosfatos/metabolismoRESUMO
Reduced nicotinamide-adenine dinucleotide oxidase of normal human polymorphonuclear leukocytes has properties that would qualify it as the enzyme responsible for the respiratory burst during phagocytosis. The enzyme was deficient in leukocytes of five patients with chronic granulomatous disease. This lack of adequate reduced nicotinamide-adenine dinucleotide oxidase could be the basis for the metabolic abnormalities characteristic of these leukocytes and for their diminished bactericidal activity.
Assuntos
Erros Inatos do Metabolismo dos Carboidratos/sangue , Neutrófilos/enzimologia , Oxirredutases/sangue , Fagocitose , Atividade Bactericida do Sangue , Doença Crônica , Cianetos , Glicólise , Hexosefosfatos/metabolismo , Humanos , Cinética , NAD , NADPRESUMO
Superoxide dismutase, catalase, glutathione peroxidase and NAD(P)H cytochrome c reductase were quantitated in polymorphonuclear leukocytes (PMN) and alveolar macrophages (AM) obtained from guinea pigs exposed up to 90 h to 85% oxygen. PMN and AM were sonicated and separated into a 16,000-g pellet, a 100,000-g pellet, and a 100,00-g supernate. Superoxide dismutase activity increased in both cells within 18 h, persisted for 66 h and decreased by 90 h. The highest rate of increase was in the 100,000-g pellet containing 3.4% of total enzyme activity in PMN but 28% in AM. The enzyme induction in PMN and AM was partially inhibited by daily intracardiac injections of 50 mg/kg actinomycin D. During oxygen exposure, catalase activity in PMN and AM decreased to 60% of its original activity, and gluthathione peroxidase was reduced in PMN to 60% and in AM to 20% of control values. Although NAD(P)H cytochrome c reductase decreased to 50% in PMN, no change was noted in AM. Upon exposure to superoxide anion, purified catalase, the glutathione peroxidase of the 100,000-g supernate, NADH, and NADPH cytochrome c reductases of the 16,000-g pellet decreased to 66+/-5%, 72+/-4%, 52+/-8%, and 40+/-9%, respectively, of their original activity. This inactivation was prevented by 0.1 mg superoxide dismutase. These in vitro observations could explain the decreased catalase and glutathione peroxidase activity demonstrated in vivo that may lead to an intracellular accumulation of hydrogen peroxide. Increased hydrogen peroxide concentrations have been found to inactivate superoxide dismutase thus impairing the first defense mechanism against superoxide anion.
Assuntos
Catalase/metabolismo , Redutases do Citocromo/metabolismo , Glutationa Peroxidase/metabolismo , Macrófagos/enzimologia , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Neutrófilos/enzimologia , Oxigênio , Peroxidases/metabolismo , Superóxido Dismutase/metabolismo , Animais , Dactinomicina , Cobaias , Modelos BiológicosRESUMO
To investigate the biochemical and cellular basis for the rise in polymorphonuclear leukocyte (PMN) count during epinephrine administration, PMN from subjects receiving epinephrine were studied for their capacity to adhere to nylon wool fibers and endothelial cell monolayers. After administration of epinephrine, the PMN count increased by 80% at 5 min, and isolated PMN adherence to nylon fibers fell from a base line of 44+/-2-18+/-3%. In contrast, when subjects were infused with the beta-antagonist propanolol before receiving epinephrine, the PMN count failed to rise and PMN adherence was normal. Exposure of PMN endothelial cell monolayers to 0.1 muM epinephrine led to diminished PMN adherence that could be blocked by 10 muM propanolol but not by 10 muM phentolamine. Sera obtained from subjects 5 min after receiving epinephrine or from supernates derived from endothelial cell monolayers exposed to 90 nM epinephrine inhibited PMN adherence to nylon fibers. Addition of anticyclic AMP antisera but not anticyclic guanosine monophosphate antisera to the postepinephrine sera or to the postepinephrine supernate derived from the endothelial cell monolayers abolished their inhibitory effect of PMN adherence to nylon fibers. In contrast, direct exposure of PMN to epinephrine failed to affect their adherent properties. Because it has been previously shown that endothelial cells contain beta-receptors and respond to catecholamines by raising their intracellular concentrations of cyclic AMP, and that PMN adherence is attenuated by cyclic AMP, it would appear that diminished PMN adherence after epinephrine administration is mediated through endothelial cell beta-receptor activity, which in turn impairs PMN margination in vivo and could account for the rise in circulating PMN.
Assuntos
Endotélio/citologia , Epinefrina/farmacologia , Neutrófilos/citologia , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores Adrenérgicos/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Endotélio/efeitos dos fármacos , Humanos , Nylons , Propranolol/farmacologiaRESUMO
A comparison has been made of the metabolic shifts in human and guinea pig leukocytes when they phagocytize. Respiration of guinea pig polymorphonuclear leukocytes (PMN) and the increment during phagocytosis were each about 2(1/2)-fold that of human PMN. This was also true of the direct oxidation of glucose-6-P (hexose monophosphate shunt). Enzymes potentially responsible for these phenomena have been compared in each species. Cyanide-insensitive NADH oxidase and NADPH oxidase were measured and only the formed exhibited adequate activity to account for the respiratory stimulus durintg phagocytosis. The hydrogen peroxide formed by this enzyme stimulates the hexose monophosphate shunt by oxidizing glutathione which upon reduction by an NADPH-linked glutathione reductase provides NADP to drive the hexose monophosphate shunt. Other linkages between respiratory stimulation and that of the hexose monophosphate shunt also pertain in the guinea pig.
Assuntos
Leucócitos/metabolismo , Consumo de Oxigênio , Animais , Glucose/metabolismo , Glutationa/metabolismo , Glutationa Redutase/metabolismo , Cobaias , Hexosefosfatos/metabolismo , Humanos , Peróxido de Hidrogênio/biossíntese , L-Lactato Desidrogenase/metabolismo , Látex/farmacologia , Leucócitos/enzimologia , Microesferas , NAD/metabolismo , NADP/metabolismo , Oxirredução , Oxirredutases/metabolismo , Fagocitose , Saponinas/farmacologia , Especificidade da EspécieRESUMO
Patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency of red blood cells (RBC) may develop sudden hemolytic anemia during infection. Since phagocytizing polymorphonuclear leukocytes (PMN) are known to generate hydrogen peroxide, we explored the influence of this oxidant product of PMN on juxtaposed G6PD-deficient and normal RBC. The oxidant stress induced by phagocytosis depleted G6PD-deficient RBC of reduced glutathione (GSH) and this was associated with rapid removal of these cells from the circulation by the liver and spleen. No such effect was observed on normal RBC. Phagocytizing chronic granulomatous disease (CGD) PMN which lack hydrogen peroxide generation, failed to diminish GSH level in G6PD-deficient RBC. Thus, PMN can pose as a source of oxidant damage to G6PD-deficient RBC due to hydrogen peroxide generated during phagocytosis.
Assuntos
Eritrócitos/enzimologia , Deficiência de Glucosefosfato Desidrogenase/sangue , Glucosefosfato Desidrogenase/metabolismo , Peróxido de Hidrogênio/metabolismo , Infecções/sangue , Leucócitos/metabolismo , Fagocitose , Anemia Hemolítica/etiologia , Células Cultivadas , Isótopos do Cromo , Eritrócitos/análise , Eritrócitos/metabolismo , Glucose Oxidase/metabolismo , Glucosefosfato Desidrogenase/análise , Deficiência de Glucosefosfato Desidrogenase/complicações , Glutationa/sangue , Glutationa/metabolismo , Corpos de Heinz/análise , Hematócrito , Hemoglobinas/análise , Humanos , Técnicas In Vitro , Infecções/complicações , Leucócitos/análise , Metemoglobina/análise , Métodos , Consumo de Oxigênio , Fatores de Tempo , Transaminases/análise , População BrancaRESUMO
The leukocytes of patients with chronic granulomatous disease (CGD) may be identified by their failure to reduce Nitro Blue Tetrazolium (NBT) during phagocytosis. This reaction, normally detected in the phagocytic vacuole, is absent or delayed in CGD monocytes and eosinophils as well as in neutrophils, even though sonicates of normal and CGD leukocytes contain equal activities of a cyanide insensitive enzyme system capable of reduction of NBT in the presence of pyridine nucleotide. Enlargement of CGD phagocytic vacuoles appears to be inhibited. Histochemical estimates of the rate of release of alkaline phosphatase are normal in CGD cells. Peroxidase activity is released from CGD cells, but the rate appears to be somewhat slower than normal in some cases. The latter observation may be explained by the increased intensity of the peroxidase stain in resting and phagocytizing CGD cells. The severity of the defect in NBT reduction within the phagocytic vacuoles of the leukocytes of patients and carriers is more variable than was previously appreciated. Some female carriers have profoundly reduced dye reduction and others are nearly indistinguishable from normal. Three brothers with CGD demonstrated significant, albeit delayed, NBT reduction in phagocytic vacuoles during prolonged incubation of their leukocytes. No obvious relationship exists, however, between the rate of reduction of NBT in vacuoles and the clinical severity of the disease.
Assuntos
Granuloma/sangue , Infecções/sangue , Leucócitos/metabolismo , Linfadenite/sangue , Fagocitose , Fosfatase Alcalina/metabolismo , Doença Crônica , Histocitoquímica , Humanos , Recém-Nascido , Doenças do Recém-Nascido , Leucócitos/enzimologia , Peroxidases/metabolismo , Sais de Tetrazólio , ZimosanRESUMO
Quantitative chemical analyses of the subcellular distribution patterns for acid and alkaline phosphatase, beta glucuronidase and peroxidase were obtained for human peripheral blood leukocytes of four patients with chronic granulomatous disease (CGD). Five young adults with acute infections served as controls. The observations were made on fractions obtained by homogenization and centrifugation of leukocytes previously incubated with or without particles for ingestion. Distributions in resting CGD and normal cells were very similar for acid and alkaline phosphatase and peroxidase, but the proportion of beta glucuronidase in the granule fraction of CGD cells was depressed, with an increased proportion in the soluble fraction. Release of granule-bound enzymes during phagocytosis of a variety of particles was the same for CGD and control cells, except that release of beta glucuronidase was less marked in CGD cells. Total enzymatic activity of CGD cells for the hydrolases studied was normal. The data indicated that granular enzymes are released in a normal fashion in phagocytizing CGD cells. Supportive evidence of release of enzymes into the phagocytic vacuole of CGD cells was obtained by an electron microscopic study of myeloperoxidase.
Assuntos
Fosfatase Ácida/metabolismo , Fosfatase Alcalina/metabolismo , Glucuronidase/metabolismo , Leucócitos/enzimologia , Peroxidases/metabolismo , Fagocitose , Histocitoquímica , Humanos , Síndromes de Imunodeficiência/genética , Microscopia EletrônicaRESUMO
D-Amino acid oxidase and L-amino acid oxidase have been measured in sucrose homogenates of polymorphonuclear leukocytes (PMN) obtained from guinea pigs and humans. Subcellular distribution patterns and studies on latency indicate that these oxidases are soluble enzymes. Their hydrogen peroxide-generating capacity was verified. Chronic granulomatous disease PMN, which lack a respiratory burst and fail to generate H(2)O(2) during phagocytosis and do not kill catalase positive bacteria, had peroxide-generating amino acid oxidase activity equal to that found in PMN homogenates from patients with bacterial infections. The precise metabolic and bactericidal role of amino acid oxidases in PMN remains uncertain.
Assuntos
Aminoácido Oxirredutases/sangue , D-Aminoácido Oxidase/sangue , Neutrófilos/enzimologia , Animais , Fracionamento Celular , Eletrodos , Feminino , Granuloma/sangue , Cobaias , Humanos , Peróxido de Hidrogênio/metabolismo , Infecções/sangue , Linfadenite/sangue , Masculino , NAD/metabolismo , Neutrófilos/metabolismo , Consumo de Oxigênio , Fagocitose , Espectrofotometria , EstereoisomerismoRESUMO
Polymorphonuclear leukocytes from patients with chronic granulomatous disease (CGD) exhibit metabolic and bactericidal deficiencies that may be the result of inadequate production of H(2)O(2). A hydrogen peroxide-generating system was, therefore, inserted into CGD leukocytes. This was accomplished by allowing the cells to phagocytize latex spherules coated with glucose oxidase. This produced an amelioration in the known metabolic deficiencies of these cells during phagocytosis: (a) intracellular (catalatic) formate oxidation dependent upon hydrogen peroxide production was enhanced fourfold; and (b) hexose monophosphate shunt activity, which other workers have shown to be at least partially dependent upon the availability of H(2)O(2), was markedly stimulated. These data strengthen the evidence that the fundamental metabolic lesion in CGD cells during phagocytosis is indeed deficient production of hydrogen peroxide, probably, as previously shown, due to diminished oxidase for reduced nicotinamide adenine dinucleotide.
Assuntos
Granuloma/sangue , Peróxido de Hidrogênio/biossíntese , Leucócitos/metabolismo , Isótopos de Carbono , Doença Crônica , Glucose Oxidase/administração & dosagem , Humanos , Peróxido de Hidrogênio/sangue , Técnicas In Vitro , Látex , Microesferas , Oxirredução , FagocitoseRESUMO
Polymorphonuclear leukocytes from humans and mice with the Chediak-Higashi syndrome were characterized by spin label electron spin resonance spectrometry. Our results suggest that cells from afflicted mice and humans have membranes more fluid than controls. Order parameters for a spin label that probes near the membrane surface were 0.652 for normals and 0.645 for two Chediak-Higashi patients. Cells from Chediak-Higashi mice showed similar differences, as did isolated plasma membrane fractions. An increased membrane fluidity was also detected with a spin label that probes deeper in the bilayer. In vitro treatment of Chediak-Higashi mouse cells with 0.01 M ascorbate increased the order parameter to normal levels. In vitro incubation of mouse Chediak-Higashi cells with glucose oxidase increased the order parameter, similar to the effect of ascorbate. This increase was abolished when catalase was added to the incubation medium. In vitro incubation with dibutyryl cyclic guanosine monophosphate (1 muM to 0.1 mM) did not normalize order parameters. These results indicate that fluidity of Chediak-Higashi cell membranes was affected by treatments expected to alter the oxidation: reduction potential of the environment but was not affected by treatments expected to alter the ratio of intracellular cyclic nucleotides. The latter treatment would affect microtubule assembly. Therefore, it appears that the membrane fluidity abnormalities as demonstrated by electron spin resonance and the earlier demonstrated microtubule dysfunctions characteristic of Chediak-Higashi cells are coexisting defects and are probably not directly related.
Assuntos
Síndrome de Chediak-Higashi/sangue , Fluidez de Membrana , Neutrófilos/metabolismo , Animais , Ácido Ascórbico/farmacologia , Membrana Celular/metabolismo , Dibutiril GMP Cíclico/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Feminino , Glucose Oxidase/farmacologia , Humanos , Peróxido de Hidrogênio/sangue , Técnicas In Vitro , Fluidez de Membrana/efeitos dos fármacos , Métodos , Camundongos , Neutrófilos/efeitos dos fármacosRESUMO
The contribution of hydrogen peroxide (H2O2) and one of its unstable intermediates, superoxide anion (O2), to the oxidative reactions that occur in phagocytizing leukocytes was explored by depleting these cells of O2. This was accomplished by allowing them to phagocytize latex particles coated with superoxide dismutase (SOD), which catalyzes the generation of H2O2 from O2. Although the rate and extent of phagocytosis of latex coated with bovine serum albumin was similar to latex coated with SOD, the rate of oxygen consumption, [14C]formate oxidation, [1-14C]glucose oxidation, and iodination of zymosan particles was significantly enhanced by SOD. In contrast, the rate and extent of reduction of nitroblue tetrazolium (NBT) was diminished by 60%. These studies indicate that the majority of NBT reduction by leukocytes is due to O2, whereas stimulation of the hexose monophosphate shunt and iodination of ingested particles requires H2O2 generated from the increased reduction of oxygen by phagocytizing leukocytes.
Assuntos
Peróxido de Hidrogênio/metabolismo , Oxigênio/metabolismo , Fagocitose , Superóxidos/metabolismo , Ânions , Formiatos/metabolismo , Glucose/metabolismo , Granulócitos/metabolismo , Humanos , Látex , Microesferas , Nitroazul de Tetrazólio/metabolismo , Oxirredução , Consumo de Oxigênio , Fagocitose/efeitos dos fármacos , Soroalbumina Bovina/metabolismo , Superóxido Dismutase/farmacologiaRESUMO
Hemoglobin (Hb) Indianapolis is an extremely labile beta-chain variant, present in such small amounts that it was undetectable by usual techniques. Clinically, it produces the phenotype of severe beta-thalassemia. Biosynthetic studies showed a beta:alpha ratio of 0.5 in reticulocytes and about 1.0 in marrow after a 1-h incubation. These results, similar to those seen in typical heterozygous beta-thalassemia, suggested that betaIndianapolis was destroyed so rapidly that its net synthesis was essentially zero. To examine the kinetics of globin synthesis, reticulocyte incubations of 1.25--20 min were performed with [3H]leucine. The betaIndianapolis:beta A ratio at 1.25 min was 0.80 suggesting that beta Indianapolis was synthesized at a near normal rate. At 20 min, this ratio was 0.46 reflecting rapid turnover of beta Indianapolis. The erythrocyte ghosts from these incubations contained only betaIndianapolis and alpha-chains, and the proportion of betaIndianapolis decreased with time, indicating loss of betaIndianapolis. Pulse-chase studies showed little change in beta A:alpha ratio and decreasing betaIndianapolis:alpha and betaIndianapolis:beta A with time. The half-life of betaIndianapolis in the soluble hemoglobin was approximately equal to 7 min. There was also rapid loss of beta Indianapolis from the erythrocyte membrane. From these results, it may be inferred that betaIndianapolis is rapidly precipitated from the soluble cell phase to the membrane, where it is catabolized. Heterozygotes for beta 0-thalassemia usually have minimal hematologic abnormalities, whereas heterozygotes with betaIndianapolis, having a similar net content of beta-chain, have severe disease. The extremely rapid precipitation and catabolism of betaIndianapolis and the resulting excess of alpha-chains, both causing membrane damage, may be responsible for the severe clinical manifestations associated with this variant. It seems likely that other, similar disturbances in the primary sequence of globin polypeptide chains may produce clinical findings similar to those seen with hemoglobin Indianapolis and thus produce the phenotype of severe beta-thalassemia.
Assuntos
Hemoglobinas Anormais/análise , Talassemia/sangue , Adolescente , Adulto , Criança , Eritrócitos/metabolismo , Família , Feminino , Globinas/biossíntese , Antígenos HLA/análise , Humanos , Ferro/sangue , Masculino , Reticulócitos/metabolismo , Talassemia/imunologiaRESUMO
Radioisotope studies of bilirubin turnover, ferrokinetics, and red cell survival ((51)Cr) in a patient with erythrocyte PK deficiency have provided evidence for prompt reticulocyte sequestration and destruction by the reticuloendothelial system. More mature erythrocytes appeared to survive well despite their deficiency of PK. PK-deficient reticulocytes, dependent upon oxidative phosphorylation for ATP production, are exquisitely sensitive to cyanide- or nitrogen-induced mitochondrial inhibition. If oxidative phosphorylation is unavailable, ATP levels decline rapidly, producing alterations in the cell membrane which allow massive losses of potassium and water. The result is a shrunken, spiculated, viscous cell whose rheologic properties would favor its sequestration by the reticuloendothelial system. Those reticulocytes with particularly low levels of PK exhibit very low glycolytic rates and thus are uniquely reliant upon oxidative phosphorylation. Other reticulocytes, better endowed with PK activity, can meet the increased ATP requirements of young erythrocytes. Upon reaching maturity, such cells have diminished ATP needs and can, therefore, survive despite their enzyme deficiency.
Assuntos
Trifosfato de Adenosina/biossíntese , Adulto , Anemia Hemolítica Congênita não Esferocítica/metabolismo , Bilirrubina/análise , Medula Óssea/análise , Criança , Pré-Escolar , Isótopos do Cromo , Cianetos/farmacologia , HumanosRESUMO
A 52 yr old Caucasian female (F. E.) had hemolytic anemia, a leukemoid reaction, and fatal sepsis due to Escherichia coli. Her leukocytes ingested bacteria normally but did not kill catalase positive Staphylococcus aureus, Escherichia coli, and Serratia marcescens. An H(2)O(2)-producing bacterium, Streptococcus faecalis, was killed normally. Granule myeloperoxidase, acid and alkaline phosphatase, and beta glucuronidase activities were normal, and these enzymes shifted normally to the phagocyte vacuole (light and electron microscopy). Intravacuolar reduction of nitroblue tetrazolium did not occur. Moreover, only minimal quantities of H(2)O(2) were generated, and the hexose monophosphate shunt (HMPS) was not stimulated during phagocytosis. These observations suggested the diagnosis of chronic granulomatous disease. However, in contrast to control and chronic granulomatous disease leukocytes, glucose-6-phosphate dehydrogenase activity was completely absent in F. E. leukocytes whereas NADH oxidase and NADPH oxidase activities were both normal. Unlike chronic granulomatous disease, methylene blue did not stimulate the hexose monophosphate shunt in F. E. cells. Thus, F. E. and chronic granulomatous disease leukocytes appear to share certain metabolic and bactericidal defects, but the metabolic basis of the abnormality differs. Chronic granulomatous disease cells lack oxidase activity which produces H(2)O(2); F. E. cells had normal levels of oxidase activity but failed to produce NADPH due to complete glucose-6-phosphate dehydrogenase deficiency. These data indicate that a complete absence of leukocyte glucose-6-phosphate dehydrogenase with defective hexose monophosphate shunt activity is associated with low H(2)O(2) production and inadequate bactericidal activity, and further suggest an important role for NADPH in the production of H(2)O(2) in human granulocytes.
Assuntos
Atividade Bactericida do Sangue , Deficiência de Glucosefosfato Desidrogenase/sangue , Leucócitos/enzimologia , Autorradiografia , Isótopos de Carbono , Ciclo do Ácido Cítrico , Enterococcus faecalis , Escherichia coli , Feminino , Granuloma/sangue , Granuloma/metabolismo , Hexosefosfatos/metabolismo , Humanos , Peróxido de Hidrogênio/biossíntese , Infecções/sangue , Infecções/metabolismo , Leucócitos/metabolismo , Linfadenite/sangue , Linfadenite/metabolismo , Microscopia Eletrônica , Pessoa de Meia-Idade , NAD/metabolismo , NADP/metabolismo , Fagocitose , Sais de Tetrazólio/metabolismoRESUMO
The capacity of human phagocytes to generate superoxide anion (O2-), a free radical of oxygen, and a possible role for this radical or its derivatives in the killing of phagocytized bacteria were explored using leukocytes from normal individuals and patients with chronic granulomatous disease (CGD). Superoxide dismutase, which removes O2-, consistently inhibited phagocytosis-associated nitroblue tetrazolium (NBT) reduction indicating the involvement of O2- in this process. Similarly, superoxide dismutase inhibited the luminescence that occurs with phagocytosis, implicating O2- in this phenomenon, perhaps through its spontaneous dismutation into singlet oxygen. Subcellular fractions from homogenates of both normal and CGD leukocytes generated O2- effectively in the presence of NADH as substrate. However, O2- generation by intact cells during phagocytosis was markedly diminished in nine patients with CGD. Leukocytes from mothers determined to be carriers of X-linked recessive CGD by intermediate phagocytic reduction of NBT elaborated O2- to an intermediate extent, further demonstrating the interrelationship between NBT reduction and O2- generation in phagocytizing cells. Activity of superoxide dismutase, the enzyme responsible for protecting the cell from the damaging effects of O2-, was approximately equal in homogenates of normal and CGD granulocytes. Polyacrylamide electrophoresis separated this activity into a minor band that appeared to be the manganese-containing superoxide dismutase associated with mitochondria and a more concentrated, cyanide-sensitive, cytosol form of the enzyme with electrophoretic mobility that corresponded to that of erythrocyte cuprozinc superoxide dismutase. Superoxide dismutase inhibited the phagocytic killing of Escherichia coli, Staphylococcus aureus, and Streptococcus viridans. A similar inhibitory effect was noted with catalase which removes hydrogen peroxide. Neither enzyme inhibited the ingestion of bacteria. Peroxide and O2- are believed to interact to generate the potent oxidant, hydroxyl radical (.OH). A requirement for .OH in the phagocytic bactericidal event might explain the apparent requirement for both O2- and H2O2 for such activity. In agreement with this possibility, benzoate and mannitol, scavengers of .OH, inhibited phagocytic bactericidal activity. Generation of singlet oxygen from O2- and .OH also might explain these findings. It would seem clear from these and other studies that the granulo cyte elaborates O2- as a concomitant of the respiratory burst that occurs with phagocytosis. To what extent the energy inherent in O2- is translated into microbialdeath through O2- itself, hydrogen peroxide, .OH, singlet oxygen, or some other agent remains to be clearly defined.
Assuntos
Doença Granulomatosa Crônica/fisiopatologia , Oxigênio , Disfunção de Fagócito Bactericida/fisiopatologia , Fagócitos/fisiologia , Superóxidos , Catalase/efeitos adversos , Grupo dos Citocromos c , Escherichia coli , Radicais Livres , Doença Granulomatosa Crônica/genética , Humanos , Leucócitos/fisiologia , Medições Luminescentes , Microscopia Eletrônica , Nitroazul de Tetrazólio , Fagocitose , Staphylococcus , Streptococcus , Superóxido Dismutase/metabolismoRESUMO
Polymorphonuclear leukocytes (PMN) aggregate and avidly attach to endothelium in response to chemotactic agents. This response may be related in part to the release of the specific granule constituent lactoferrin (LF). We found by using immunohistology and biochemical and biophysical techniques that LF binds to the membrane and alters the surface properties of the PMN. Upon exposure of PMN treated with 5 micrograms/ml cytochalasin B to 2 x 10(-7) M formyl-methionine-leucine-phenylalanine for 5 min, the PMN mobilized LF to their surface as observed by immunoperoxidase staining for LF. At added LF levels ranging from 4 to 15 micrograms/10(7) PMN there was a dose-dependent reduction in PMN surface charge reaching 4 mV, when the partitioning into the membrane of a charged amphipathic nitroxide spin label was measured by electron spin resonance spectroscopy, whereas transferrin was without effect. When 125I-FeLF was added to human PMN in increasing amounts and the results corrected for the residual amount of free LF contaminating the cells, the PMN were saturated with LF at concentrations between 100 and 200 nM in the medium. Human PMN bound 1.35 x 10(6) molecules per cell and the calculated value for the association constant for these receptors was 5.2 x 10(6) M-1. Additionally, 6 micrograms/ml LF served as an opsonin for rabbit MN to promote PMN uptake by rabbit macrophages, when assessed by electron microscopy, but lysozyme did not. These studies indicate that LF can bind to the surface of the PMN and reduce its surface charge. This correlates with enhanced "stickiness" leading to a variety of cell-cell interactions.
Assuntos
Lactoferrina/metabolismo , Lactoglobulinas/metabolismo , Neutrófilos/metabolismo , Animais , Sítios de Ligação , Adesão Celular/efeitos dos fármacos , Comunicação Celular , Membrana Celular/metabolismo , Citocalasina B/farmacologia , Grânulos Citoplasmáticos/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , N-Formilmetionina/análogos & derivados , N-Formilmetionina/farmacologia , N-Formilmetionina Leucil-Fenilalanina , Oligopeptídeos/farmacologia , Coelhos , Propriedades de SuperfícieRESUMO
Pleural fluid from a child previously treated for rhabdomyosarcoma produced colonies in vitro. Cells from these colonies appeared to have the light and electron microscopic appearance of rhabdomyosarcoma cells. In this case, the malignant nature of the effusion had been suspected because of the patient's previous history; however, this technique may prove useful in the diagnosis of effusion of unknown etiology.
Assuntos
Técnicas de Cultura , Derrame Pleural/patologia , Rabdomiossarcoma/patologia , Animais , Núcleo Celular/ultraestrutura , Criança , Citoplasma/ultraestrutura , Cães , Retículo Endoplasmático/ultraestrutura , Feminino , Complexo de Golgi/ultraestrutura , Humanos , Mitocôndrias/ultraestruturaRESUMO
Leukemic monoblasts obtained from three children were evaluated for their in vitro physiologic characteristics. These monoblasts were alpha naphthyl acetate esterase positive, exhibited glass adherence, had IgG membrane receptors, phagocytized latex particles with subsequent nitroblue tetrazolium (NBT) reduction, and matured to macrophages in vitro, either when adherent to glass slides or in feeder layer suspensions. Employing the double layer in vitro bone marrow culture technique, leukemic marrows from these children failed to produce colonies in culture in the presence or absence of normal feeder layers. Varying the concentration of leukemic cells in the feeder layers failed to augment colony growth from normal bone marrows. Although monoblasts were observed to mature to macrophages in vitro, no increase in colony stimulating factor activity was observed with aging of the feeder layers. These studies suggest that childhood leukemic monoblasts lack the ability to produce colony stimulating activity, differing in this respect from monoblastic leukemia in adults.