RESUMEN
We investigated reactive oxygen species (ROS) involvement in polymorphonuclear neutrophilic leukocyte (neutrophil) apoptosis triggering. Neutrophils were incubated with xanthine oxidase (XO), which produces superoxide anion (O2.-) and hydrogen peroxide (H2O2) or glucose oxidase (GO), which produces only H2O2. Both XO and GO accelerated apoptosis when compared to spontaneously aged neutrophils. Catalase inhibited both spontaneous apoptosis and XO- or GO-accelerated apoptosis, but superoxide dismutase did not. Hydrogen peroxide can enter the cell, thus generating intracellular oxidation, which was observed by flow cytometry. Furthermore, the intracellular reduced glutathione content fell in the presence of XO or GO; however, apoptosis was not accelerated in the presence of buthionine sulfoximine (BSO), suggesting that the fall in glutathione in the presence of XO or GO is a consequence of oxidative stress but not a trigger of apoptosis. Hydrogen peroxide can react with iron to form hydroxyl radicals (HO.); we observed that two iron chelators, deferoxamine and hydroxybenzyl ethylenediamine (HBED), both inhibited spontaneous and accelerated apoptosis, suggesting that HO. may mediate neutrophil apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Radical Hidroxilo/farmacología , Neutrófilos/fisiología , Catalasa/farmacología , Fragmentación del ADN , Citometría de Flujo , Glucosa Oxidasa/metabolismo , Glutatión/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Radical Hidroxilo/metabolismo , Hierro/metabolismo , Quelantes del Hierro/farmacología , Oxidación-Reducción , Superóxido Dismutasa/farmacología , Superóxidos/metabolismo , Xantina Oxidasa/metabolismoRESUMEN
Hydrogen peroxide (H2O2) increases adherence of human polymorphonuclear neutrophils (PMN) to cultured human umbilical vein endothelial cells (HUVEC). Catalase and HO. scavengers did not affect the increased PMN adherence to HUVEC stimulated by other compounds such as phorbol myristate acetate (PMA) and thrombin, showing that the observed effect was H2O2- and HO.-specific. This effect was inhibited by hydroxyl radicals (HO.) scavengers and not by iron-chelators that do not penetrate the cells, suggesting the involvement of intracellular HO. in the increased adherence mechanism. An increase in cAMP inhibited H2O2-induced adherence, as observed with isoproterenol, isobutylmethylxanthine, and dibutyryl-cAMP. Similarly, pentoxifylline (Ptx), an HO. scavenger that also increases cAMP, inhibited H2O2-mediated adherence but had no effect on that induced by PMA or thrombin. PKA inhibitors cancelled the Ptx-induced inhibition of H2O2-mediated adherence. However, PKA inhibitors or atrial natriuretic peptide that decreases cAMP did not increase adherence, showing that decrease in cAMP is not responsible for increased adherence. HO. scavengers did not alter the H2O2-induced reduction in cAMP levels, but did inhibit the effect of H2O2 on adherence. We conclude that HO. mediates the H2O2-induced increased in PMN adherence to HUVEC, and that the increase in cAMP that mediates PKA activation downregulates this effect.
Asunto(s)
Adhesión Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Endotelio Vascular/fisiología , Depuradores de Radicales Libres/farmacología , Peróxido de Hidrógeno/farmacología , Radical Hidroxilo , Neutrófilos/fisiología , Pentoxifilina/farmacología , 1-Metil-3-Isobutilxantina/farmacología , Análisis de Varianza , Bucladesina/farmacología , Adhesión Celular/fisiología , Células Cultivadas , Deferoxamina/farmacología , Glutatión/metabolismo , Humanos , Hipoxantina/farmacología , Isoproterenol/farmacología , Neutrófilos/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Trombina/farmacología , Transferrina/farmacología , Venas Umbilicales , Xantina Oxidasa/farmacologíaRESUMEN
Rats exposed to normobaric oxygen received a single i.p. injection of 2.5 mg/kg of poly I: poly C at various times (-120 to +36 h) before and after the beginning of oxygen exposure. Hyperoxic lung damage and modifications in cytochrome P-450 system components were evaluated. Our results confirmed the protective effect of poly I: poly C on rats exposed to oxygen, reducing the lung edema and the mortality. This effect was only observed when poly I: poly C was injected 48 or 36 h before the beginning of oxygen exposure. Although oxygen exposure per se decreased the total level of lung cytochrome P-450, poly I: poly C per se induced a deeper decrease to levels similar in air- or oxygen-exposed rats. Poly I: poly C did not modify the NADPH-cytochrome c reductase level nor the cytochrome P-450 peroxidase activity in air-exposed rats. The oxygen exposure induced a decrease of these two enzymes, either in the absence or in the presence of poly I: poly C, except when poly I: poly C was injected 48 or 36 h before the beginning of oxygen exposure, times at which poly I: poly C restored the enzymatic values measured in rats exposed to air. Because the times of injection of poly I: poly C were those at which the protective effect was observed, it suggested that the protective effect of poly I: poly C against oxygen toxicity was associated with a lack of oxygen-induced decrease of both the lung NADPH-cytochrome c reductase level and the lung cytochrome P-450 peroxidase activity.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Pulmón/enzimología , NADPH-Ferrihemoproteína Reductasa/metabolismo , Oxígeno/efectos adversos , Peroxidasas/metabolismo , Poli I-C/uso terapéutico , Animales , Glutatión Peroxidasa/efectos de los fármacos , Glutatión Reductasa/efectos de los fármacos , Pulmón/efectos de los fármacos , Masculino , Derrame Pleural/prevención & control , Edema Pulmonar/prevención & control , Ratas , Superóxido Dismutasa/efectos de los fármacos , Factores de TiempoRESUMEN
Phorbol esters such as phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBU) are generally considered to have similar effects through a similar mechanism, i.e. protein kinase C (PKC) activation. We recently suggested that this was not the case in human neutrophils. To identify further differences between the two phorbol esters, we compared their priming effects on fMet-Leu-Phe-induced superoxide anion (O2-) production, cytosolic PKC activity and binding of fMet-Leu-Phe. Priming could be initiated with a low (0.2 nM) concentration of both PDBU and PMA. Their effects on the pattern of fMet-Leu-Phe-induced superoxide production were similar in both Ca2(+)-containing and Ca2(+)-free medium. PDBU, like PMA, abolished the Ca2+ dependency of fMet-Leu-Phe-induced O2- production in a dose-dependent manner. In cytochalasin B-treated cells and in the presence of Ca2+, priming with PDBU or PMA did not alter the enhancing effect of cytochalasin B on fMet-Leu-Phe-induced O2- production. In Ca2(+)-free medium, priming abolished the Ca2+ dependency of fMet-Leu-Phe stimulation in cytochalasin B-treated cells. Cytochalasin B, however, enhanced the effect of PMA but not that of PDBU. Priming with PDBU was not associated under any experimental conditions with a decrease in cytosolic PKC activity, or an increase in PKM activity before or after fMet-Leu-Phe stimulation. Furthermore, priming effects were abolished by cell washing but not by H-7 or staurosporine, which are potent PKC inhibitors. PDBU, in contrast to PMA, increased fMet-Leu-Phe binding to PMNs through a decrease in the dissociation constant and induced degranulation of specific granules as measured by the release of vitamin B12 binding protein. These findings show that the priming effects of PDBU differ in certain respects from those of PMA, namely with regard to its synergism with cytochalasin B and the expression of fMet-Leu-Phe receptors. In addition, priming concentrations of PDBU, like PMA, did not alter cytosolic PKC activity in fMet-Leu-Phe-stimulated neutrophils.
Asunto(s)
N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Forbol 12,13-Dibutirato/farmacología , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Células Cultivadas , Citocalasinas/farmacología , Humanos , Neutrófilos/metabolismo , Proteína Quinasa C/análisisRESUMEN
Phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBU) are known to translocate protein kinase C (PKC) and to induce superoxide anion (O2-.) production in human neutrophils. They are thus currently used to probe the role of PKC in O2-. production. We show here that under certain conditions, O2-. production induced by PMA is not associated with a decrease in cytosolic PKC activity, whereas these two events are associated after PDBU stimulation. (1) In the presence of extracellular calcium (1 mM), O2-. production was related to the concentration of PMA. PMA induced O2-. production at all the concentrations studied, but this was not associated with a decrease in cytosolic PKC levels up to 5 ng/ml PMA (50% maximum O2-. production). (2) Under PDBU stimulation, even at very low O2-. production levels, cytosolic PKC decreased and the decrease as well as the O2-. production were related to the concentration of PDBU. (3) For a given decrease in cytosolic PKC, O2-. production induced by PMA was much greater than that induced by PDBU. (4) In calcium-free medium, O2-. production induced by low concentrations of PMA (up to 5 ng/ml) was lower than that observed in the presence of 1 mM calcium, whereas modifications of cytosolic PKC activity were similar. (5) Cytochalasin B had no effect on PMA-induced O2-. production, regardless of the calcium content of the medium, and had no effect on the decrease in cytosolic PKC. On the contrary, following PDBU stimulation, cytochalasin B increased O2-. production, regardless of the medium, but induced a larger decrease in cytosolic PKC when Ca2+ was present. (6) Preincubation of PMN with 100 microM H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) before stimulation with PMA or PDBU led to similar inhibition of O2-. production whatever the degree of decrease in cytosolic PKC activity. These findings show that, in contrast to PDBU, O2-. production induced by PMA is not always related to cytosolic PKC activity.
Asunto(s)
Neutrófilos/metabolismo , Forbol 12,13-Dibutirato/farmacología , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Calcio/metabolismo , Cloruro de Calcio/farmacología , Citocalasina B/farmacología , Citosol/efectos de los fármacos , Citosol/enzimología , Ácido Edético/farmacología , Humanos , Técnicas In Vitro , Indicadores y Reactivos , Isoquinolinas/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Piperazinas/farmacología , Proteína Quinasa C/metabolismoRESUMEN
Phorbol myristate acetate (PMA) is a potent activator of Ca2+/phospholipid-dependent protein kinase (PKC) and was used to study the involvement of this kinase in human polymorphonuclear neutrophil (PMN) locomotion. Preincubation of PMNs with low concentrations of PMA (4 to 64 x 10(-11) M) had the following effects: (1) fMet-Leu-Phe-induced migration under agarose was increased when the chemoattractant was used at the suboptimal concentration of 10(-8)M and not at the optimal concentration of 10(-7)M; (2) no effect on spontaneous or serum- or LTB4- induced migration at either suboptimal or optimal concentrations; (3) PMA enhanced fMet-Leu-Phe-induced migration, increasing the speed of locomotion but not affecting shape changes induced by fMet-Leu-Phe; (4) the number of fMet-Leu-Phe-specific receptors expressed on the PMN membrane was not altered. Intermediate concentrations of PMA (1.6 to 4.0 x 10(-9) M) had no effect on PMN migration, whereas higher concentrations (4.0 to 16 x 10(-9) M) reduced both spontaneous and fMet-Leu-Phe-, serum-, or LTB4-induced migration in a dose-dependent manner. Effects observed with low concentrations of PMA were not associated with a translocation of cytosolic PKC even when preincubation with PMA was followed by fMet-Leu-Phe stimulation. In contrast, effects observed with higher concentrations of PMA paralleled the decrease in cytosolic PKC activity.
Asunto(s)
Quimiotaxis de Leucocito/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Células Cultivadas , Citosol/enzimología , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Humanos , Leucotrieno B4/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/fisiología , Neutrófilos/ultraestructura , Proteína Quinasa C/metabolismo , Receptores de Formil Péptido , Receptores Inmunológicos/efectos de los fármacosRESUMEN
Whether or not cytochrome b-559 is a necessary component of NADPH oxidase activity in neutrophils is still controversial. In highly purified plasma membranes isolated from resting neutrophils and lacking cytochrome b, addition of arachidonic acid induced an NADPH oxidase activity. This activity was similar to that of plasma membranes isolated from phorbol myristate acetate (PMA)-stimulated cells which possessed cytochrome b. Addition of arachidonic acid to the latter plasma membranes did not alter the oxidase activity. It can be concluded that plasma membranes isolated from resting neutrophils have, in the presence of arachidonic acid, an NADPH oxidase activity similar to that of PMA-stimulated cells, except that it is independent of cytochrome b-559.
Asunto(s)
Ácidos Araquidónicos/sangre , Grupo Citocromo b/sangre , NADH NADPH Oxidorreductasas/sangre , Neutrófilos/fisiología , Complejo de Proteína del Fotosistema II , Ácido Araquidónico , Membrana Celular/enzimología , Activación Enzimática , Humanos , NADPH Oxidasas , Superóxidos/metabolismoRESUMEN
The effects of the nonsteroidal antiinflammatory drug indomethacin on the parameters relating to the migration and respiratory burst of human polymorphonuclear leukocytes (PMN) were studied in an attempt to clarify the mechanism of this drug's action on PMN. At various concentrations below 200 micrograms/ml, indomethacin partially inhibited the spontaneous migration of PMN but did not alter the directional migration induced by C5a-activated serum. In the presence of N-formyl-methionyl-leucyl-phenylalanine (FMLP) as chemoattractant, directed PMN migration was either inhibited or stimulated by indomethacin, depending on FMLP concentration. When PMN migration was induced by the optimal and suboptimal FMLP concentrations of 10(-7) and 10(-8) M, indomethacin inhibited this migration, but when the high FMLP concentration of 10(-6) M depressed this migration by chemotactic deactivation, indomethacin restored it to its maximum. Both the inhibitory and stimulatory effects of indomethacin on FMLP-induced PMN migration were due to changes in the migration speed. Indomethacin also inhibited FMLP-induced changes in the shape of floating PMN, and in respiratory burst, as well as specific FMLP binding to PMN. In contrast, indomethacin did not alter the PMN respiratory burst induced by phorbol myristate acetate or C5a-activated serum. These data show that indomethacin is able to prevent the loss of PMN chemokinetic activity induced by formylated peptides and suggest that it might be useful for investigating the mechanism of peptide-induced chemotactic deactivation.
Asunto(s)
Quimiotaxis de Leucocito/efectos de los fármacos , Indometacina/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In Vitro , Neutrófilos/citología , Neutrófilos/fisiología , Consumo de Oxígeno/efectos de los fármacos , Receptores de Formil Péptido , Receptores Inmunológicos/metabolismo , Superóxidos/metabolismoRESUMEN
The effects of 1-(5-isoquinoline sulphonyl)-2-methyl piperazine (H-7), a recently described inhibitor of Ca2+/phospholipid-dependent protein kinase (protein kinase C), were studied during under-agarose migration of human polymorphonuclear neutrophils (PMN) stimulated by various chemoattractants, in order to determine whether protein kinase C is involved in PMN locomotion. The effect of H-7 on the oxidative burst induced by phorbol 12-myristate 13-acetate or N-formyl-methionyl-leucyl-phenylalanine (FMLP) was also measured. Pre-incubation of PMN with H-7 concentrations ranging from 50 to 400 microM inhibited: (i) spontaneous PMN migration under agarose; (ii) the directed migration induced by activated serum, leukotriene B4 or FMLP; and (iii) the speed of the migration induced by FMLP. The inhibition by H-7 of FMLP-induced directed migration was less when FMLP was used at high concentrations which, in the absence of H-7, inhibit locomotion. H-7 depressed the oxidative burst induced by phorbol myristate acetate (PMA) but not that induced by FMLP. All the effects of H-7 on the oxidative burst and migration were reversed by washing PMN after H-7 treatment. These findings indicate that protein kinase C, inhibitable by H-7, is involved in a mechanism controlling the speed of PMN locomotion.
Asunto(s)
Quimiotaxis de Leucocito/efectos de los fármacos , Isoquinolinas/farmacología , Neutrófilos/fisiología , Piperazinas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Adulto , Movimiento Celular/efectos de los fármacos , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/metabolismo , Superóxidos/sangreRESUMEN
The effects of cytochrome P-450 inducers on O2 toxicity were studied in mice. We first examined three cytochrome P-450 inducers, which differ by their specific tissue affinity: phenobarbital sodium (PB), essentially active in the liver, and 3-methylcholanthrene (3-MC) and beta-naphthoflavone (BNF), which are also active in the lung. Both BNF and 3-MC increased the survival rate and significantly decreased pulmonary edema (pulmonary water and wet-to-dry weight ratio) in C57BL/6J mice exposed to hyperoxia (O2 greater than or equal to 95%), whereas PB had no protective effect. In the second part of this study, we compared the action of BNF in two strains of mice. In one (C57BL/6J), cytochrome P-450 can be induced by aromatic hydrocarbons, whereas in the other (DBA/2J) cytochrome P-450 is not inducible by these compounds. Protection against O2 toxicity was assessed in terms of lethality and pulmonary edema and of lung lipid peroxidation (assessed by measuring malondialdehyde). BNF only protected against O2 toxicity in the inducible strain. This protective effect of BNF on O2 toxicity in C57BL/6J mice was associated mainly with a large increase in the components of the cytochrome P-450 system (cytochrome P-450 and cytochrome b5) in the lung. The activity of pulmonary superoxide dismutase was also slightly increased, but the enhancement was not statistically significant. In contrast, in DBA/2J mice neither the components of the cytochrome P-450 system nor the activity of superoxide dismutase showed any increase.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Benzoflavonas/farmacología , Sistema Enzimático del Citocromo P-450/biosíntesis , Flavonoides/farmacología , Metilcolantreno/farmacología , Microsomas Hepáticos/metabolismo , Oxígeno/toxicidad , Fenobarbital/farmacología , Edema Pulmonar/prevención & control , Animales , Inducción Enzimática , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Microsomas Hepáticos/efectos de los fármacos , Edema Pulmonar/etiología , Edema Pulmonar/genética , Especificidad de la Especie , beta-naftoflavonaRESUMEN
Rats were pretreated with various inducers of cytochrome P-450 before being exposed to pure normobaric oxygen (O2) in order to determine whether the inducers interfere with toxicity. The pulmonary and liver inducers beta-naphthoflavone (beta NF) and 3-methylcholanthrene (3MC) increased the survival rate and decreased the amount of pleural and lung fluid accumulation in adult rats exposed to oxygen. Phenobarbital (PB), which is essentially active in the hepatic microsomal cytochrome P-450, was less effective in counteracting oxygen toxicity. After 7 days of exposure to oxygen, none of the untreated rats survived, whereas 40, 73, and 90% survival was observed in rats treated with PB, 3MC, and beta NF, respectively. After 60 h of O2 exposure, significantly less pleural and lung fluid accumulation was observed in beta NF- and 3MC-treated rats than in untreated or PB-treated rats (p less than 0.001). Both beta NF and 3MC prevented the increase of lung peroxidation (assessed by measuring of malondialdehyde) and that of hydrogen peroxide production by lung microsomes induced by O2 exposure. These protective effects are associated with a large increase in the components of the pulmonary cytochrome P-450 system and its peroxidase activity and with an increased response to hyperoxia by lung antioxidant enzyme activities. In contrast, in control rats, the activities of the antioxidant enzymes were not increased, and both the quantity and the peroxidase activity of cytochrome P-450 were significantly decreased by O2 exposure. We conclude that in the rat, pretreatment by inducers of pulmonary cytochrome P-450 results in marked protection against O2 toxicity and an increase of antioxidant enzyme response to hyperoxia.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Sistema Enzimático del Citocromo P-450/fisiología , Oxígeno/toxicidad , Edema Pulmonar/etiología , Animales , Benzoflavonas/farmacología , Peróxido de Hidrógeno/biosíntesis , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Malondialdehído/metabolismo , Metilcolantreno/farmacología , Microsomas/metabolismo , Fenobarbital/farmacología , Edema Pulmonar/tratamiento farmacológico , Edema Pulmonar/fisiopatología , Distribución Aleatoria , Ratas , Ratas Endogámicas , Superóxidos/biosíntesis , beta-naftoflavonaRESUMEN
In order to analyse the role of LFA1 and MO1 on neutrophil functions, the blocking effects of two monoclonal antibodies (MAb), one (anti-MO1) recognizing an epitope of the MO1-alpha chain and the other (25.31) an epitope of the LFA1-alpha chain, were measured. Adherence of 51Cr-labelled control neutrophils was 66 + 8% (mean +/- 1 SD) on plastic nuclon plates; this figure decreased to 33 +/- 5% and 23 +/- 6% of control adherence when the neutrophils had been pretreated with anti-LFA1-alpha (anti-alpha L) and anti-MO1-alpha (anti-alpha M), respectively. On another support (plastic culture chambers), 84 +/- 6% of control neutrophils adhered and the adherence of neutrophils pretreated with anti-alpha L or anti-alpha M was 10% and 43% of the control figure, respectively. These results show that adherence of neutrophils is dependent upon the plastic used. Moreover, inhibition of adhesion by the two MAbs was also dependent upon the support used for the assay, suggesting that MO1 and LFA1 may be surface proteins with different specificities. Both antigens capped upon adhesion, while they were randomly distributed in resting neutrophils. Anti-alpha L inhibited (congruent to 50%) locomotion more than did anti-alpha M (congruent to 25%), without altering chemoattractant-induced shape changes. These results suggest that the two MAbs inhibit chemokinesis but not chemotaxis. Many other adherence-associated functions, such as ingestion of opsonized Klebsiella pneumoniae, and cytotoxicity towards K/562 cells were decreased more by anti-alpha L than by anti-alpha M. In contrast, chemiluminescence and iodination induced by opsonized zymosan were inhibited more by anti-alpha M than by anti-alpha L. Degranulation induced by zymosan or opsonized zymosan was altered by anti-alpha M only, and this alteration involved azurophilic and not specific granules. Chemiluminescence induced by phorbol myristate acetate was inhibited to a greater extent by anti-alpha M than by anti-alpha L, while degranulation induced by phorbol myristate acetate was not altered by either of the two Mabs.
Asunto(s)
Antígenos de Superficie/inmunología , Neutrófilos/inmunología , Receptores de Complemento/inmunología , Adulto , Anticuerpos Monoclonales/inmunología , Unión Competitiva , Adhesión Celular , Movimiento Celular , Citotoxicidad Inmunológica , Humanos , Antígeno-1 Asociado a Función de Linfocito , Neutrófilos/fisiología , Fagocitosis , Receptores de Complemento 3bRESUMEN
Prenatal diagnosis of chronic granulomatous disease (CGD) was performed in four male high risk fetuses. The male sex was previously determined by an amniotic cell karyotype. Three kinds of test were performed on fetal blood obtained by umbilical venous puncture under fetoscopy at the 20th gestational week: nitroblue tetrazolium reduction (NBT) cytochemical test with phorbol myristate acetate (PMA) as activator; luminol enhanced chemiluminescence with activation by serum opsonized zymosan (STZ) or PMA; superoxide anion (0-2) production by measurement of the superoxide dismutase inhibitable reduction of cytochrome c with PMA as activator. Results were compared to those obtained in six fetuses investigated for other inherited diseases. In one case, absence of granulocyte defects was confirmed at birth. In three other cases, the tests showed deficient metabolic oxidative granulocytes. The pregnancy was terminated and the CGD diagnosis was confirmed on the products of abortion. The use of three different techniques performed on whole blood for CGD prenatal diagnosis is recommended instead of a single isolated test to ensure a higher confidence in the diagnosis.
Asunto(s)
Enfermedades Fetales/diagnóstico , Granulocitos/metabolismo , Enfermedad Granulomatosa Crónica/diagnóstico , Diagnóstico Prenatal , Femenino , Sangre Fetal , Humanos , Masculino , Oxidación-Reducción , Embarazo , RiesgoRESUMEN
Plasma membranes of high purity and good yield have been prepared from human polymorphonuclear neutrophils by a one-step procedure involving disruption of cells suspended in paraffin oil and forced by pressure through an annular slit. This results in a band floating above the oil which is composed of large sheets of plasma membranes. Enrichment values for the plasma membrane marker alkaline phosphatase and 125I-labeled protein after surface labeling performed at the whole cell level were 23-fold and 22-fold, respectively. Contamination of the plasma membrane by other organelles was negligible and approximately 2 mg of membrane protein was obtained from 10(9) neutrophils. The procedure is very fast and the use of paraffin oil avoids lengthy high-speed centrifugation. The technique also allows isolation of granules devoid of plasma membrane and can probably be applied to other cell types.
Asunto(s)
Fraccionamiento Celular/métodos , Neutrófilos/ultraestructura , Aceites , Adulto , Membrana Celular/enzimología , Citoplasma/análisis , Humanos , Proteínas de la Membrana/sangre , Neutrófilos/análisis , Parafina , Fracciones Subcelulares/análisisRESUMEN
Resting neutrophils possess cytosolic cyanide-sensitive (CNs) superoxide dismutase (SOD) and cyanide-insensitive (CNi) SOD, located in an undefined organelle of the 27,000 g sedimentable fraction of its homogenate. Stimulated neutrophils generate large amounts of superoxide anion, part of which is released in the extracellular medium and contributes to changes that occur in inflammatory foci. Our purpose was to assess whether or not the neutrophil upon stimulation secreted either or both CNs and CNi SOD activity, because the process could protect against the release of superoxide anion. Human neutrophils stimulated in vitro with phorbol myristate acetate released 32.6% and 53% of their content in myeloperoxidase (an azurophilic granule marker) and vitamin B12 binding activity, respectively. The CNi SOD was not secreted at all, whereas 16% and 23% of CNs SOD were released by resting and stimulated neutrophils, respectively. In contrast, lactate dehydrogenase, a cytosolic marker, was released by both resting and stimulated cells (approximately equal to 9%). These results suggest that CNi SOD is not located in the granules but in another organelle that does not degranulate upon stimulation and consequently does not protect against superoxide anion formed by neutrophils in the extracellular medium. In contrast, CNs SOD is slightly but significantly released (P less than .02) and may be protective. Neutrophils from two patients with chronic granulomatous disease behaved similarly to control neutrophils but their content of both types of SOD was higher than that of the controls.
Asunto(s)
Neutrófilos/enzimología , Superóxido Dismutasa/sangre , Citocalasina B/farmacología , Gránulos Citoplasmáticos/enzimología , Enfermedad Granulomatosa Crónica/enzimología , Humanos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
To kill microorganisms, phagocytes exhibit an oxidative burst with, in particular, a NADPH-dependent, superoxide-generating system that consists, in polymorphonuclear leukocytes (PMN), of a flavin enzyme and cytochrome b-245 (cyt b-245). We investigated the existence of this cytochrome in human alveolar macrophages (AM) because its presence would support its wide-spread occurrence in phagocytes and would raise the possibility of similarities in the oxygen-dependent killing mechanisms in AM and PMN. Moreover, we compared the amount of cyt b-245 in AM from patients with lung disorders with that from healthy subjects, by a differential spectroscopic measurement of its 558 to 559 nm characteristic band. This spectrum showed that cyt b-245 was present in AM. In AM of healthy subjects, the amount was similar to that found in PMN of blood. In AM of patients with miscellaneous lung diseases and in Am of infected lungs, the data were not modified.
Asunto(s)
Grupo Citocromo b/metabolismo , Macrófagos/metabolismo , Alveolos Pulmonares/metabolismo , Histiocitosis de Células de Langerhans/metabolismo , Humanos , Infecciones/metabolismo , Enfermedades Pulmonares/metabolismo , Linfocitos/metabolismo , Neutrófilos/metabolismo , Alveolos Pulmonares/citología , Alveolos Pulmonares/patología , Fibrosis Pulmonar/metabolismo , Sarcoidosis/metabolismoRESUMEN
In vivo exposure of mice to normobaric O2 depresses the cellular immune response by a mechanism that remains unknown. In vitro oxidative injury leads to decreased sulfhydryl groups (SH) in lymphocytes. To determine whether in vivo exposure to O2 would have similar effects, we measured the SH content in spleen cells both from mice that had been exposed to normobaric O2 (O2 SC) and from controls exposed to ambient air (Air SC). The SH content of the fresh O2 SC was slightly decreased, whereas after 48 hr of culture, the SH content and the proliferative response of these cells were found to vary with the type and concentration of thiol or disulfide compounds added to the culture medium. Under standard culture conditions, i.e., RPMI 1640 medium containing 0.41 mM half-cystine, the SH content in O2 SC decreased sharply to about 10 and 20% that of Air SC in the absence or presence of Con A (2 micrograms/ml), respectively. Under these culture conditions, the proliferative response of O2 SC was 20.5% +/- 3.2 of Air SC. In cystine-free RPMI 1640 medium supplemented with various concentrations of L-cystine, L-cystine and 2-mercaptoethanol (2-ME), L-cysteine, or reduced glutathione (GSH), the proliferative response to Con A and the SH content of the O2 SC varied in parallel and were correlated (p less than 0.01). Half-cystine (0.41 mM) plus 2-ME (5 X 10(-5) M) or L-cysteine alone (4 mM) completely protected the SH content of O2 SC and induced a proliferative response 82% +/- 6 that of the controls. In cystine-free RPMI 1640 medium supplemented with GSH (4 mM), the SH content and proliferative response of O2 SC were 79 and 67.5% of Air SC, respectively. Other concentrations of these compounds were less effective. Oxygen scavengers such as SOD, catalase, mannitol, and vitamin E did not protect against the decrease of the O2 SC. The induced oxidative cellular damage might be related in part to a membrane lipid peroxidative process. These data show that in vivo exposure of mice to normobaric O2 induced lesions in splenic cells manifested under standard culture conditions by a decrease in both SH content and Con A proliferative response. The extent of these alterations could be modulated by variations of the thiol environment. Protection of the SH content correlated with protection of the proliferative response of the O2 SC.
Asunto(s)
Activación de Linfocitos/efectos de los fármacos , Oxígeno/toxicidad , Compuestos de Sulfhidrilo/farmacología , Animales , Células Cultivadas , Concanavalina A/farmacología , Cisteína/fisiología , Femenino , Radicales Libres , Glutatión/farmacología , Glutatión/fisiología , Peróxidos Lipídicos/metabolismo , Mercaptoetanol/farmacología , Ratones , Ratones Endogámicos C57BL , Receptores de Concanavalina A/metabolismo , Compuestos de Sulfhidrilo/fisiologíaRESUMEN
A cytochrome b, designated as cytochrome b-245, exists in neutrophils and is probably involved in their stimulated oxidative burst. As a rule, its concentration is spectroscopically measured by the height of its alpha-peak at 558-559 nm (dithionite-reduced minus oxidized). Hemoglobin (Hb), which usually contaminates neutrophils isolated from blood, interferes with the spectroscopic measurement of the cytochrome. Hb contamination from less than 0.4 red blood cells per 100 neutrophils leads to over-estimation of the cytochrome by approximately 50%. This interference can be overcome by bubbling CO through neutrophil homogenates heavily contaminated by Hb, prior to the conventional spectroscopic procedure. The cytochrome B-245 concentration obtained in neutrophils by CO bubbling is 7.2 +/- 1.28 pmoles per 10(6) polymorphonuclear neutrophils.
Asunto(s)
Grupo Citocromo b/análisis , Neutrófilos/análisis , Monóxido de Carbono , Grupo Citocromo b/sangre , Enfermedad Granulomatosa Crónica/sangre , Hemoglobinas/análisis , Humanos , EspectrofotometríaRESUMEN
A microtechnique for determining the superoxide dismutase activity in erythrocytes is described. This technique involves the inhibition of luminol-enhanced chemiluminescence of superoxide anion generated by xanthine-xanthine oxidase. Measurements required a steady-state chemiluminescence whether superoxide dismutase was present or absent; the level of luminescence was correlated to enzyme activity. Superoxide dismutase activity measured by this technique was 836 +/- 112 micrograms/g of hemoglobin for whole blood and 834 +/- 109 micrograms/g of hemoglobin for erythrocytes. When the reference technique was applied to larger amounts of blood, the results were 862 +/- 58 and 858 +/- 116 micrograms/g of hemoglobin for whole blood and washed erythrocytes, respectively. The enzymatic activity of superoxide dismutase from fetal blood (obtained by venipuncture in utero and of 19-26 weeks gestational age) was similar to that of adult blood, when measured by the new technique.
Asunto(s)
Sangre Fetal/enzimología , Superóxido Dismutasa/sangre , Adulto , Eritrocitos/enzimología , Femenino , Humanos , Cinética , Mediciones Luminiscentes , Luminol , Microquímica , Embarazo , Xantina , Xantina Oxidasa , XantinasRESUMEN
Latex ingestion and nitroblue tetrazolium reduction were measured in neutrophils of 40 patients with primary idiopathic myelofibrosis. The percentage of neutrophils that ingested latex, in the presence of either autologous or control sera, was lower (P less than 0.001) than that of the controls. The percentage of the ingesting neutrophils that reduced nitroblue tetrazolium was also lower (P less than 0.001) in the patients than in the controls. Activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione peroxidase and NAD(P)H oxidases were not different from those of the controls. In contrast, glutathione reductase activity was significantly lower (P less than 0.001) in the patients than in the controls either with or without the addition of flavin adenine dinucleotide. Glutathione reductase and nitroblue tetrazolium reduction activities were correlated (r = 0.913). These results are discussed within the framework of the acquired enzymopathies and the increased susceptibility to infection observed in these patients.