RESUMO
We studied the chemotactic peptide receptor/cytoskeletal interactions in HL-60 cells induced to differentiate with different agents and attempted to correlate these observations with the acquisition of different functional responses. Dibutyryl cyclic AMP-treated cells showed rapid superoxide anion production in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) and slow, sustained response to phorbol myristate acetate (PMA). Retinoic acid-induced cells showed a slow, sustained response to both FMLP and PMA. Interferon-gamma-treated cells produced no superoxide anion on stimulation with FMLP, whereas tumor necrosis factor (TNF)-treated cells showed a slight response. Chemotactic peptide receptor association was the same in the HL-60 cells treated with different agents, despite marked differences in the superoxide anion generation and actin polymerization responses to FMLP and PMA in these cells. In mature neutrophils chemotactic peptide receptor association with the cytoskeleton was not affected by either pertussis or cholera toxin. However, both toxins inhibited FMLP-induced actin polymerization and superoxide anion generation. This suggested involvement of a G-protein similar to Gt, rather than Gi or Gs. Neither toxin had any effect on PMA-induced superoxide anion generation. These observations indicate that receptor association with the cytoskeleton may not have a significant role in affecting signal recognition and response. Among the several possible roles suggested, clearance of the occupied receptors may be the most important role of the cytoskeletal association. HL-60 cells induced to differentiate with different agents (because of their varied functional responses) might prove very useful in dissecting the molecular mechanisms regulating stimulus-induced activation of neutrophils.
Assuntos
Citoesqueleto/fisiologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/fisiologia , Receptores Imunológicos/fisiologia , Bucladesina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Toxina da Cólera/farmacologia , Citoesqueleto/ultraestrutura , Interferon gama/farmacologia , Leucemia Mieloide/patologia , Receptores de Formil Peptídeo , Superóxidos/biossíntese , Tretinoína/farmacologia , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Xanthine oxidase activity in the rat brain was measured by means of high-performance liquid chromatography with electrochemical detection of uric acid. Cerebral ischemia was produced by a four-vessel occlusion method. In the control rat, the enzyme activity was 0.87 +/- 0.13 nmol/gm wet weight/min at 25 degrees C (mean +/- standard deviation), of which 92.4% was associated with the nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase form and only 7.6% with the oxygen-dependent superoxide-producing oxidase form. However, the ratio of the latter form increased to 43.7% after 30 minutes of global ischemia, despite the total xanthine oxidase activity remaining the same. Thus, it was revealed that uric acid can be synthesized in the rat brain and that cerebral ischemia induced the conversion of xanthine oxidase from an NAD-dependent dehydrogenase to an oxygen-dependent superoxide-producing oxidase. Although the xanthine oxidase pathway has been proposed as a source of oxygen-derived free radicals in various ischemic organs other than brain, the results of the present study suggest the involvement of the oxygen free radicals generated from this pathway in the pathogenesis of the ischemic injury of the rat brain.
Assuntos
Isquemia Encefálica/enzimologia , Encéfalo/enzimologia , Xantina Oxidase/metabolismo , Animais , Isoenzimas/metabolismo , Masculino , NAD/farmacologia , Oxigênio/farmacologia , Ratos , Ratos Endogâmicos , Superóxidos/biossíntese , Xantina Desidrogenase/metabolismoRESUMO
Although prior studies with mAb have defined an endogenous chymotrypsin-like protease in the neutrophil (polymorphonuclear leukocyte (PMN)) membrane that is associated with initiation of superoxide response to inflammatory stimuli, it is not known whether extracellular proteases (in the inflammatory milieu) can also influence PMN activation. This study examined the ability of four neutral proteases: cathepsin G, elastase, chymotrypsin, and trypsin, to modify PMN superoxide response to FMLP, PMA, and arachidonate. In response to 1 microM FMLP, PMN treated with cathepsin G, chymotrypsin, or elastase showed 64%, 60%, and 32% increases, respectively, in superoxide generation when compared with control, untreated cells (p less than 0.05 for each). These increments were dependent on intact enzymatic function of the proteases, were greatest when enzyme and stimulus were added concurrently, and persisted after PMN were washed free of enzyme. Enhancement of superoxide response was not stimulus specific; in response to 10 ng/ml PMA, cells treated with cathepsin G showed a 84%, and elastase a 57%, increase in superoxide generation (p less than 0.05 for both) with a marked reduction in the time required for onset of this response. For cell activation with 80 microM arachidonate, treatment with elastase produced a 180% increase in superoxide production (p less than 0.025). Neutrophils incubated with trypsin demonstrated significant decreases in superoxide response to PMA (-34%, p less than 0.05) and arachidonate (-39%, p less than 0.01). The enzymes themselves were not stimuli for superoxide production nor were they scavengers for superoxide in cellfree system. We conclude that local release of the PMN primary-granule neutral proteases, cathepsin G, and elastase within inflammatory sites can augment neutrophil effector function by up-regulating oxidative response to defined inflammatory stimuli. This autocrine/paracrine function may provide a significant increase in antimicrobial activity, but may also enhance the potential for host tissue injury.
Assuntos
Neutrófilos/metabolismo , Peptídeo Hidrolases/farmacologia , Superóxidos/biossíntese , Adulto , Ácido Araquidônico , Ácidos Araquidônicos/farmacologia , Catepsina G , Catepsinas/farmacologia , Quimotripsina/farmacologia , Combinação de Medicamentos , Humanos , Cinética , Muramidase/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/enzimologia , Elastase Pancreática/farmacologia , Serina Endopeptidases , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
We used a monoclonal antibody, YI 51, raised against human polymorphonuclear leukocytes (PMN) to induce superoxide anion (O2-) generation in cells. Although YI 51 alone played only a small part in inducing O2- generation in PMN, the amount of O2- generation induced in 5 X 10(5) PMN was 3.7 to 5.5 nmol/min when F(ab')2 fragments of rabbit anti-mouse immunoglobulin antibody were added as a cross-linking agent. This O2- -inducing activity was high compared with that of wheat germ agglutinin (WGA), insoluble immunoglobulin G immune complexes (IC), or phorbol myristate acetate (PMA). The binding of YI 51 and soluble immunoglobulin G IC to PMN was not reciprocally inhibitory, indicating that YI 51 does not interfere with ligand binding to the Fc receptor-binding site. In the absence of calcium ion (Ca2+), O2- generation induced by YI 51 decreased to 10 to 20% of that in the presence of Ca2+. In contrast, O2- generation in response to WGA, IC, or PMA under Ca2+-free conditions was not affected. When PMN were pretreated with low concentrations of PMA (10(-10) to 10(-9) M), the amount of O2- generation by the cells in response to YI 51 in Ca2+-free buffer was enhanced in a concentration-dependent manner. It also equaled the O2- generated by the cells in buffer containing Ca2+. In cells pretreated with PMA, the amount of O2- induced by WGA was enhanced two- to threefold over that in untreated cells. In contrast, there was no augmentation over untreated cells with stimulation by IC. These results suggest that YI 51, IC, and WGA induce O2- generation in human PMN in different manners.
Assuntos
Anticorpos Monoclonais/fisiologia , Cálcio/fisiologia , Neutrófilos/metabolismo , Superóxidos/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , Animais , Anticorpos Monoclonais/biossíntese , Complexo Antígeno-Anticorpo/metabolismo , Antígenos de Diferenciação/metabolismo , Sítios de Ligação de Anticorpos/efeitos dos fármacos , Espaço Extracelular/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Receptores Fc/análise , Receptores Fc/metabolismo , Receptores de IgG , Receptores Mitogênicos/efeitos dos fármacos , Aglutininas do Germe de Trigo/metabolismoRESUMO
Although zinc is essential for the optimum function of the immune system, there is some controversy regarding treatment with zinc during acute infections where low serum zinc levels are often recorded. The aim of the present study was to investigate the influence of in vitro and in vivo zinc supplementation on the potentially toxic metabolic activity of peritoneal macrophages during infection. Rats were made septic by implanting a gelatin capsule containing known amounts of E. coli, and Bacteroides fragilis into the abdomen. Peritoneal macrophages were harvested by peritoneal lavage 72 hours after the induction of sepsis. Superoxide release was measured after stimulation with phorbol myristate acetate (PMA) or serum treated zymosan (STZ). Macrophages from septic rats released significantly higher amounts of superoxide compared with macrophages from sham operated controls after stimulation with both PMA and STZ. Following in vitro supplementation, zinc inhibited the superoxide production of macrophages harvested from septic rats after stimulation with both PMA and STZ. In vivo supplementation with zinc resulted in increased superoxide production from septic macrophages when stimulated with STZ, whereas stimulation with PMA produced no significant changes. Thus, in vitro incubation inhibited the superoxide production of peritoneal macrophages in intraabdominal sepsis, whilst in vivo administration of zinc produced no such effect, and the effect seemed to vary depending on the stimuli used to initiate the respiratory burst.
Assuntos
Macrófagos/metabolismo , Superóxidos/biossíntese , Zinco/farmacologia , Animais , Infecções por Bacteroides/metabolismo , Bacteroides fragilis , Infecções por Escherichia coli/metabolismo , Técnicas In Vitro , Macrófagos/efeitos dos fármacos , Masculino , Cavidade Peritoneal/efeitos dos fármacos , Cavidade Peritoneal/metabolismo , Ratos , Ratos EndogâmicosRESUMO
Byssinosis is a hazardous respiratory disorder of workers in natural fiber processing industries and, in the case of cotton, is caused by exposure to respirable dust generated from leafy trash associated with raw fibers. To understand the chemical characteristics of involucral trash components that might contribute to bysinosis, we examined the human airway constricting activity and oxygen radical generating activity of dry, frost-killed cotton bracts. In response to inhalation of aerosolized bract extracts, the expiratory flow rates of human volunteers at 40% of vital capacity during partial forced expiration decreased by 3 to 32%. These values enabled us to identify two potentially byssinogenically active bract specimens, a specimen virtually inactive, and a fourth intermediately so. Using spin trapping techniques of electron spin resonance spectrometry, we found that all specimens catalyzed the generation of hydroxyl (preponderantly) and superoxide radicals from hydrogen peroxide. However, the weakest constrictor was the most potent catalyst, and vice versa. This was consistent with transition metal content of the specimens; the most potent catalyst also contained the largest amounts of those metals, suggesting a Fenton-type reaction mechanisms. Other possibilities for the inverse relationship of airway constricting (byssinogenic) activity with oxygen radical generation are discussed. We also found that neither aflatoxin nor endotoxin, contingent contaminants of bracts, catalyzed oxygen radical production from hydrogen peroxide.
Assuntos
Bissinose/etiologia , Gossypium/metabolismo , Aflatoxina B1 , Aflatoxinas/farmacologia , Bissinose/fisiopatologia , Óxidos N-Cíclicos , Espectroscopia de Ressonância de Spin Eletrônica , Endotoxinas/farmacologia , Fluxo Expiratório Forçado , Volume Expiratório Forçado , Radicais Livres , Gossypium/análise , Gossypium/toxicidade , Humanos , Peróxido de Hidrogênio/metabolismo , Hidróxidos/metabolismo , Radical Hidroxila , Minerais/análise , Marcadores de Spin , Superóxidos/biossínteseRESUMO
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine which produces diverse biological effects in target cells of myeloid origin. GM-CSF enhances the production of superoxide anion (O2-) by mature neutrophils in response to chemotactic peptides such as formyl-methionyl-leucyl-phenylalanine (fMLP), but alone it has no effect on this system. This process has been termed "priming." fMLP activates neutrophils via a pertussis toxin-sensitive GTP-binding protein, leading to the rapid production of the second messengers diacylglycerol (DAG) and inositol trisphosphate, via phosphatidylinositol turnover, and arachidonic acid (AA) by a presumptive phospholipase A2-mediated mechanism. All three second messengers may lead to the generation of O2-. We investigated the effect of priming of GM-CSF on these systems. GM-CSF had no effect on fMLP-stimulated DAG and inositol trisphosphate levels, nor did it amplify the response to exogenously added phorbol ester (to mimic the action of DAG) or calcium ionophore. Neutrophils primed with the cytokine showed a small, but significant, enhancement of fMLP-stimulated AA release. Compared with unprimed controls, primed neutrophils also showed a significant increase in O2- production when stimulated with either AA or the nonhydrolyzable GTP analogue, GTP-gamma-S. The magnitude of enhanced O2- production was similar to that observed after fMLP treatment of primed cells. All of these effects, including the increased sensitivity to AA treatment, were inhibited by pertussis toxin. These data show that GM-CSF primes neutrophils by modulating the activity of at least one pertussis toxin-sensitive G protein coupled to a metabolic pathway that mobilizes and utilizes arachidonic acid.
Assuntos
Fatores Estimuladores de Colônias/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Substâncias de Crescimento/farmacologia , Neutrófilos/fisiologia , Toxina Pertussis , Fosfatidilinositóis/metabolismo , Fatores de Virulência de Bordetella , Adulto , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Ligação Competitiva , Cálcio/fisiologia , Proteínas de Ligação ao GTP/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Humanos , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidases , Neutrófilos/metabolismo , Proteína Quinase C/farmacologia , Proteínas Recombinantes/farmacologia , Superóxidos/biossínteseRESUMO
Crystals and a group of ceramics used in joint replacement surgery were incubated with polymorphonuclear cells (PMN) with and without adsorbed immunoglobulin G (IgG). All 11 particles tested had the capacity to induce the production of superoxide anion (O2-) to at least some extent. Synthetic calcium pyrophosphate dihydrate crystals (CPPD) and several ceramics adsorbed small amounts of IgG and the increment of superoxide production was also small. Both the augmentation of O2- and the adsorption of IgG to hydroxyapatite type I synthetic crystals and the remaining ceramics were intermediate. Synthetic monosodium urate crystals adsorbed the most IgG and the O2- increments were large. The increase in superoxide production correlated with the amount of IgG bound onto crystals or ceramics. The greater protein binding and phlogistic potential of some ceramics needs to be taken into consideration in their clinical use.
Assuntos
Cerâmica , Imunoglobulina G/farmacologia , Neutrófilos/metabolismo , Superóxidos/biossíntese , Ácido Úrico/farmacologia , Adsorção , Fosfatase Alcalina , Cristalização , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoglobulina G/farmacocinética , Oxigênio/biossíntese , Tamanho da PartículaRESUMO
The effects of ATP on the concentration of cytosolic calcium [( Ca2+]i) were examined with respect to early events associated with activation of the superoxide (O2-)-generating system in human neutrophils. Addition of ATP to cytochalasin B-treated neutrophils resulted in two sequential increase in [Ca2+]i: an initial phase presumably related to the mobilization of Ca2+ from intracellular stores and a second phase dependent upon the presence of extracellular Ca2+. The second phase was associated with an increase in the rate of O2- production, which also required the presence of extracellular Ca2+. The results suggest that increased Ca2+ influx may act to trigger a cascade of Ca2+-sensitive events, leading to stimulated O2-production.
Assuntos
Trifosfato de Adenosina/farmacologia , Fura-2/análogos & derivados , Neutrófilos/metabolismo , Superóxidos/biossíntese , Benzofuranos , Cálcio/sangue , Citocalasina B , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/fisiologia , Corantes Fluorescentes , Humanos , Cinética , Neutrófilos/efeitos dos fármacos , Superóxidos/sangueRESUMO
We have compared platelet activating factor (PAF)-induced eosinophil peroxidase (EPO) release and intracellular calcium mobilization with superoxide anion (.O2-) generation from guinea pig eosinophils. EPO release and Ca2+ mobilization occurred at lower concentrations of PAF (EC50 values of 1.3 nM and 11.5 nM, respectively) while .O2- production was observed at higher concentrations (EC50 of 31.7 microM). Receptor characterization with the competitive PAF antagonist, WEB 2086, gave pA2 values of 8.5 and 8.3 for EPO enzyme release and rise in [Ca2+]i, respectively, and 5.8 for the .O2- production. In addition, PAF-induced degranulation and elevation of [Ca2+]i were dependent on extracellular Ca2+ whereas PAF-stimulated .O2- generation was dependent on the presence of extracellular Mg2+ ions. These results suggest the existence either of two subtypes of the PAF receptor or a single receptor that can exist in one of two affinity states on guinea pig eosinophils.
Assuntos
Cálcio/fisiologia , Eosinófilos/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas , Receptores de Superfície Celular/análise , Receptores Acoplados a Proteínas G , Superóxidos/biossíntese , Triazóis , Animais , Azepinas/farmacologia , Cálcio/biossíntese , Cálcio/farmacologia , Peroxidase de Eosinófilo , Eosinófilos/efeitos dos fármacos , Eosinófilos/fisiologia , Cobaias , Magnésio/farmacologia , Masculino , Peroxidases/metabolismo , Fator de Ativação de Plaquetas/antagonistas & inibidores , Receptores de Superfície Celular/fisiologia , Transdução de Sinais/efeitos dos fármacos , Triazinas/farmacologiaRESUMO
Superoxide anion production by polymorphonuclear leukocytes stimulated with phorbol 12-myristate 13-acetate is known to be inhibited by a number of inhibitors and substrates of serine proteases, in particular by tosylphenylalanylchloromethane (TosPheCH2Cl) and to a lesser extent by tosyllysylchloromethane (TosLysCH2Cl). We have reinvestigated the characteristics of this inhibition, in view of the fact that other serine protease inhibitors with similar specificities, phenylmethanesulfonyl fluoride and leupeptin, were without effect. We found that the inhibition of phorbol-ester-induced superoxide production after cell preincubation with the chloromethanes followed saturation kinetics, with Kinact and kinact values of 100 microM and 31 min-1 for TosPheCH2Cl and 2 mM and 18 min-1 for TosLysCh2Cl. We also showed that the two compounds, which can inhibit protein kinase C in vitro, inhibited neither its activity in vivo, nor its translocation induced by phorbol myristate acetate. Furthermore the intracellular non-protein sulfhydryl group content was not affected by the treatment with the chloromethanes. Finally, addition of the inhibitors to stimulated cells also led to a time-dependent, concentration-dependent inhibition of superoxide production. Altogether, our results suggest that the chloromethane target is neither a protease nor protein kinase C and is not involved in NADPH oxidase activation, but rather in maintenance of its activity. The possible identity of this protein is discussed.
Assuntos
Clorometilcetonas de Aminoácidos/farmacologia , NADH NADPH Oxirredutases/antagonistas & inibidores , Neutrófilos/enzimologia , Inibidores de Proteases/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , NADPH Oxidases , Neutrófilos/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Proteína Quinase C/antagonistas & inibidores , Superóxidos/biossíntese , Tosilina Clorometil Cetona/farmacologiaRESUMO
Both the chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP) and the calcium ionophore ionomycin induced a metabolic response in normal neutrophils. However, the presence of azide, a potent inhibitor of the hydrogen peroxide-consuming enzymes catalase and myeloperoxidase, was required to detect any release of hydrogen peroxide induced by ionomycin. In differentiated HL-60 cells, only FMLP stimulation was associated with any notable metabolic activation. The response to FMLP proceeds with a rate and time course similar to that seen in normal cells. The use of ionomycin as a stimulating agent did not result in any detectable activation of the system that generates reactive oxygen metabolites, even if azide was present in the measuring system. Raising the concentration of cytoplasmic free Ca2+ is therefore not sufficient to activate the system responsible for the generation of reactive oxygen metabolites in HL-60 cells. However, preincubation with ionomycin primed HL-60 cells to an increased response during stimulation with the chemotactic peptide FMLP and the phorbol ester PMA. Since HL-60 cells lack specific granules but have an intact ligand-receptor coupling mechanism, a role for the subcellular granule is proposed, in the generation of reactive oxygen species in normal granulocytes, and analysis of the data presented leads to two conclusions: 1) FMLP, which acts through cells surface receptors, causes the cells to produce oxygen radicals, which to a large extent are released from the cells, a process that is not dependent on the specific granule content of the cells, whereas 2) ionomycin, which bypasses cell-surface receptors, is also capable of stimulating an oxygen-radical formation that is granule dependent and retained inside the cells. Furthermore, the results suggest that an increase in intracellular Ca2+ is not sufficient to initiate activation of the plasma membrane-bound system that generates reactive oxygen metabolites, but the results support a role for Ca2+ in the priming event.
Assuntos
Ionóforos/farmacologia , Neutrófilos/efeitos dos fármacos , Oxigênio/sangue , Adulto , Cálcio/sangue , Éteres/farmacologia , Humanos , Peróxido de Hidrogênio/biossíntese , Ionomicina , Medições Luminescentes , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADH NADPH Oxirredutases/sangue , NADPH Oxidases , Neutrófilos/metabolismo , Superóxidos/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
The in vitro generation and release of 5-lipoxygenase metabolites of arachidonic acid by bovine peripheral blood neutrophils and alveolar neutrophils elicited with either a heat-killed bacterium, Haemophilus somnus, or platelet-activating factor, were compared. After stimulation with calcium ionophore A23187 for 2.5-60 min, up to 4.5 +/- 0.7 (mean +/- SEM) ng of LTB4 per 10(6) cells was released into the media by circulating neutrophils. LTB4 release by alveolar neutrophils was significantly less (P less than .05) than that of peripheral blood neutrophils from the same animal; 5-HETE release by circulating neutrophils was maximal after 5 min stimulation by ionophore (1.2 +/- 0.1 ng/10(6) cells) but was not identified in cell culture media after 20 min. Alveolar neutrophils released similar amounts of 5-HETE when compared to circulating neutrophils, and release of 5-HETE by alveolar neutrophils was maximal after 5 min of stimulation. However, the 5-HETE released into the culture media persisted throughout the 60 min time period at levels which were maximal (1.5 +/- 0.2 to 1.8 +/- 0.3 ng/10(6) cells). Bacterial killing and the release of superoxide anion were not different between the two cell populations.
Assuntos
Ácidos Araquidônicos/metabolismo , Atividade Bactericida do Sangue , Neutrófilos/metabolismo , Alvéolos Pulmonares/citologia , Superóxidos/biossíntese , Animais , Ácido Araquidônico , Bovinos , Haemophilus , Ácidos Hidroxieicosatetraenoicos/metabolismo , Leucotrieno B4/metabolismo , Masculino , Neutrófilos/fisiologia , Fator de Ativação de Plaquetas , Staphylococcus epidermidisRESUMO
Modulation of neutrophil activation by catecholamines reflects a fine-tuning by coupling inhibitory and stimulatory receptor pathways. The catecholamine isoproterenol (ISO) binds to beta-adrenergic cell surface receptors and thereby inhibits cell responses such as O2- production stimulated by formyl peptides. However, ISO did not inhibit O2- generation activated by 1 microM ionophore A23187, the protein kinase C activators phorbol ester (PMA, 100 ng/ml) and oleoylacetylglycerol (OAG, 50 microM), and the G-protein activator NaF (40 mM). Furthermore, the overall kinetics of oxidant production in the presence of ISO were unchanged when cells were stimulated with PMA, OAG, A23187, and NaF. These results would imply that neither intracellular calcium, the activation of protein kinase C, nor the activation of G-protein are the primary target of the inhibitory pathway. Accordingly, pertussis toxin did not block PMA or NaF-stimulated superoxide generation. In contrast, formyl peptide-dependent GTPase activity is inhibited by ISO in sonicated cell preparations. Since ISO increases the cAMP concentration in the cell, the possibility is raised that a cAMP-dependent kinase inhibits signal transduction in part by blocking the interaction of this receptor with its G-protein.
Assuntos
Isoproterenol/farmacologia , Neutrófilos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais , AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Toxina Pertussis , Fosforilação , Proteína Quinase C/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Superóxidos/biossíntese , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Glucocorticoids inhibit superoxide (O2-) generation by phagocytes through a mechanism that remains unclear. We investigated this effect by using dexamethasone on guinea pig alveolar macrophages. O2- generation was induced either by the calcium ionophore A23187, a potent stimulus of phospholipase A2, or by the protein kinase C activator, phorbol myristate acetate (PMA). Dexamethasone inhibited O2- generation initiated by A23187 by 50-55%. This inhibition required: (a) more than 45 min incubation and was maximal after 2 h; (b) glucocorticoid receptor occupancy; and (c) protein synthesis. The inhibitory effect of dexamethasone could not be explained by an interaction with the respiratory burst enzyme NADPH oxidase since O2- generation was only weakly affected upon PMA stimulation. Lipocortin I, a glucocorticoid inducible and phospholipase A2 inhibitory protein, inhibited O2- generation initiated by A23187 but failed to modulate the respiratory burst activated by PMA. These results were obtained with lipocortin I purified from mouse lungs, human blood mononuclear cells, and with human recombinant lipocortin I. We propose that lipocortin I is capable of inhibiting the activation of NADPH oxidase only when membrane signal transduction involves phospholipase A2. By mimicking the effect of dexamethasone, lipocortin I may extend its potential anti-inflammatory action to the partial control of the formation of oxygen reactive species by phagocytes.
Assuntos
Dexametasona/farmacologia , Glicoproteínas/farmacologia , Macrófagos/metabolismo , Superóxidos/antagonistas & inibidores , Animais , Anexinas , Calcimicina/farmacologia , Dinoprostona/biossíntese , Cobaias , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidases , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2 , Fator de Ativação de Plaquetas/farmacologia , Alvéolos Pulmonares , Proteínas Recombinantes/farmacologia , Superóxidos/biossínteseRESUMO
Cultured porcine retinal pigment epithelial cells release superoxide, measured as superoxide dismutase (SOD)-suppressible reduction of cytochrome C, and SOD-suppressible luminol-dependent chemiluminescence. Latex beads stimulated a significant release of superoxide that reached 82 nmol/mg protein in the first 15 min and declined thereafter. Formation of an insoluble blue formazan following reduction of nitroblue tetrazolium histochemically demonstrated that superoxide was released over the region of the cell vicinal to the bead. Dioctanoylglycerol, a synthetic, cell-permeating activator of protein kinase C, also elicited a rapid release of superoxide from RPE cells. This study emphasizes the need to characterize the mechanisms by which superoxide is generated, whether its release is regulated, and how toxicity is prevented in vivo.
Assuntos
Epitélio Pigmentado Ocular/metabolismo , Superóxidos/biossíntese , Animais , Ácidos Araquidônicos/farmacologia , Células Cultivadas , Diglicerídeos/farmacologia , Microesferas , Nitroazul de Tetrazólio , Epitélio Pigmentado Ocular/citologia , Superóxido Dismutase/farmacologia , Suínos , Acetato de Tetradecanoilforbol/farmacologia , Fatores de TempoRESUMO
Interleukin-1 (IL-1) plays a major role in the response to infection, inflammation, and immunological challenge. Eosinophils participate in the host response to parasitic infection and allergic and hypersensitivity diseases. The role of IL-1 in these disease states has not been extensively explored. We have reported that purified human monocyte derived IL-1 (mIL-1), a mixture of the two IL-1 forms but predominantly consisting of IL-1 beta, modulates eosinophil oxidative metabolism and enzyme secretion. Although the two major species of IL-1 (IL-1 alpha and IL-1 beta) have identical specific activities on T cells, we now report the selective effects of human recombinant IL-1 (hrIL-1) alpha and hrIL-1 beta on eosinophil function. Whereas hrIL-1 beta caused a significant increase in arylsulfatase secretion (235.4 +/- 29% of resting secretion, P less than or equal to .01) and beta-glucuronidase secretion (135.8 +/- 9.6% of resting secretion, P less than or equal to .02) similar to our experience with mIL-1, hrIL-1 alpha had no effect on enzyme secretion. However, a mixture of hrIL-1 alpha and hrIL-1 beta reproduced the ability of mIL-1 to inhibit the oxidative response to suboptimal doses of phorbol myristate acetate (PMA). When eosinophils were separated into subpopulations by density gradients, we found that eosinophil responses to IL-1 differed among the populations. These results suggest that eosinophil subpopulations respond selectively to each form of IL-1.
Assuntos
Eosinófilos/efeitos dos fármacos , Interleucina-1/farmacologia , Arilsulfatases/sangue , Separação Celular , Centrifugação com Gradiente de Concentração , Grânulos Citoplasmáticos/enzimologia , Eosinófilos/enzimologia , Eosinófilos/metabolismo , Glucuronidase/sangue , Humanos , Interleucina-1/metabolismo , Receptores Imunológicos/análise , Receptores de Interleucina-1 , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Relação Estrutura-Atividade , Superóxidos/biossínteseRESUMO
Airway inflammation with neutrophil infiltration may play a role in airway hyperreactivity. Neutrophils may exert their effects through the generation of superoxide O2- anion and other oxygen-derived free radicals. O2- generation by neutrophils has been demonstrated to be modulated by adenosine at physiologic concentrations. Therefore, we have investigated the function of peripheral blood neutrophils with respect to O2- anion generation and its regulation by adenosine in both subjects with asthma and normal subjects and also the relationship between O2- anion generation and airway hyperresponsiveness in subjects with asthma. Purified neutrophils were obtained from eight subjects with stable asthma and seven normal control subjects not taking chronic medications. O2- anion generation in subjects with asthma was significantly higher compared with that of normal subjects after stimulation with either N-formyl-methionyl-leucyl-phenylalanine (mean, 14.8 nmol/10(6) cells for subjects with asthma versus mean, 9.6 nmol/10(6) cells for normal subjects; p less than 0.01) or phorbol myristate acetate (mean, 13.6 nmol/10(6) cells versus mean, 8.1 nmol/10(6) cells; p less than 0.05). Adenosine inhibited N-formyl-methionyl-leucyl-phenylalanine-stimulated O2- anion generation in a dose-related fashion in subjects with asthma and normal subjects to a similar degree. Adenosine had no effect on O2- anion generation after phorbol myristate acetate stimulation. These results indicate that neutrophils from subjects with asthma produce more O2- anion when they are stimulated than do neutrophils from normal subjects and that this difference is not due to adenosine modulation. In subjects with asthma, O2- anion generation correlated with the degree of airway hyperresponsiveness to inhaled methacholine.
Assuntos
Adenosina/farmacologia , Asma/sangue , Neutrófilos/metabolismo , Superóxidos/biossíntese , Administração por Inalação , Adolescente , Adulto , Feminino , Humanos , Masculino , Cloreto de Metacolina , Compostos de Metacolina/administração & dosagem , Pessoa de Meia-Idade , N-Formilmetionina Leucil-Fenilalanina , Neutrófilos/efeitos dos fármacos , Superóxidos/sangueRESUMO
We investigated the ability of the lymphokine, interleukin-4 (IL-4), to function as a neutrophil (PMN) activator. IL-4 enhanced PMN-mediated killing of opsonized bacteria (by up to 91.6% at 3 units of IL-4; p less than 0.05). IL-4 was a weak secondary granule secretagogue and did not by itself generate a respiratory burst. However, IL-4 did increase in a dose-dependent fashion the respiratory burst mediated by the peptide formyl-methionyl-leucyl-phenylalanine (10(-7) mol/L). Maximal potentiation of PMN activity occurred at 100 units of IL-4 (6.3 nmol superoxide produced without IL-4 to 9.8 nmol at 100 units; p less than 0.01). Enhancement of the respiratory burst was not a generalized phenomenon, since IL-4 did not potentiate the respiratory burst mediated by either phorbol myristate acetate, calcium ionophore A23187, or zymosan-treated serum. Similarly, IL-4 potentiated the formyl-methionyl-leucyl-phenylalanine-stimulated secretion of both lysozyme (40.2%) and beta-glucuronidase (108.2%). Finally, IL-4 was demonstrated to enhance the ability of PMN to phagocytose sheep erythrocytes opsonized with rabbit IgG (by up to 94.2% at 30 units of IL-4). This increased phagocytosis correlated with the recruitment of a population of PMNs that did not phagocytose targets in the absence of IL-4. In conclusion, IL-4 enhanced neutrophil-mediated bactericidal activity. This increase may have occurred secondary to the stimulation of phagocytosis by IL-4 or by potentiation of degranulation and the respiratory burst.
Assuntos
Interleucinas/fisiologia , Neutrófilos/fisiologia , Atividade Bactericida do Sangue , Grânulos Citoplasmáticos/enzimologia , Glucuronidase/sangue , Humanos , Técnicas In Vitro , Interleucina-4 , L-Lactato Desidrogenase/sangue , Ativação Linfocitária , Muramidase/sangue , Neutrófilos/enzimologia , Fagocitose , Superóxidos/biossíntese , Superóxidos/sangueRESUMO
To examine the modulatory effects of catecholamines on the respiratory burst in polymorphonuclear leukocytes (PMNs), dopamine was tested for its capacity to modify the superoxide anion (O2-) production by PMNs under their stimulation with several stimuli. Dopamine inhibited the O2- production by PMNs when PMNs were stimulated with N-formylated chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP), phorbol myristate acetate, or opsonized zymosan, whereas dopamine did not alter the PMN mobility. The values of percentage inhibition of the O2- production by FMLP-stimulated PMNs were 57% under treatment with 10(-5) mol/L and 83% with 10(-4) mol/L of dopamine. Isoproterenol also inhibited PMN O2- production in response to FMLP. Although a beta-adrenergic blockade, propranolol, diminished the isoproterenol-induced inhibition of the O2- production, it did not affect the inhibitory effect of dopamine. The increase in intracellular cyclic AMP levels in dopamine-treated PMNs was much smaller than the increase in isoproterenol-treated cells. Furthermore, dopamine inhibited the reduced nicotinamide-adenine dinucleotide phosphate-dependent O2- production by subcellular particles. These results indicate that dopamine inhibits PMN O2- production through its effect on PMN reduced nicotinamide-adenine dinucleotide phosphate-oxidase system rather than through its beta-adrenergic action.