RESUMO
The primary objective of the current study was to investigate the potential of the pneumococcal toxin, pneumolysin (Ply), to activate neutrophil extracellular trap (NET) formation in vitro. Isolated human blood neutrophils were exposed to recombinant Ply (5-20 ng ml(-1) ) for 30-90 min at 37°C and NET formation measured using the following procedures to detect extracellular DNA: (i) flow cytometry using Vybrant® DyeCycle™ Ruby; (ii) spectrofluorimetry using the fluorophore, Sytox(®) Orange (5 µM); and (iii) NanoDrop(®) technology. These procedures were complemented by fluorescence microscopy using 4', 6-diamino-2-phenylindole (DAPI) (nuclear stain) in combination with anti-citrullinated histone monoclonal antibodies to visualize nets. Exposure of neutrophils to Ply resulted in relatively rapid (detected within 30-60 min), statistically significant (P < 0·05) dose- and time-related increases in the release of cellular DNA impregnated with both citrullinated histone and myeloperoxidase. Microscopy revealed that NETosis appeared to be restricted to a subpopulation of neutrophils, the numbers of NET-forming cells in the control and Ply-treated systems (10 and 20 ng ml(-1) ) were 4·3 (4·2), 14.3 (9·9) and 16·5 (7·5), respectively (n = 4, P < 0·0001 for comparison of the control with both Ply-treated systems). Ply-induced NETosis occurred in the setting of retention of cell viability, and apparent lack of involvement of reactive oxygen species and Toll-like receptor 4. In conclusion, Ply induces vital NETosis in human neutrophils, a process which may either contribute to host defence or worsen disease severity, depending on the intensity of the inflammatory response during pneumococcal infection.
Assuntos
DNA/imunologia , Armadilhas Extracelulares/imunologia , Neutrófilos/efeitos dos fármacos , Estreptolisinas/farmacologia , Anticorpos Monoclonais/química , Proteínas de Bactérias/farmacologia , Sobrevivência Celular , Citrulina/imunologia , DNA/agonistas , DNA/metabolismo , Expressão Gênica , Histonas/genética , Histonas/imunologia , Humanos , Indóis , Neutrófilos/citologia , Neutrófilos/imunologia , Peroxidase/genética , Peroxidase/imunologia , Cultura Primária de Células , Espécies Reativas de Oxigênio/imunologia , Proteínas Recombinantes/farmacologia , Streptococcus pneumoniae/química , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
INTRODUCTION: Despite the high burden of disease, there have been surprisingly few studies of the acute phase and plasma catecholamine/cortisol stress hormone responses in patients with active pulmonary tuberculosis. We wished to document acute phase reactant and stress hormone responses in patients with newly diagnosed, active pulmonary tuberculosis and to compare these responses to those of a group of surgical/medical cases with conditions other than tuberculosis. METHODS: This was a prospective study of consecutive patients with newly diagnosed pulmonary tuberculosis, admitted to a tertiary hospital in Johannesburg, South Africa, documenting demographic, clinical, routine laboratory, acute phase protein and stress hormone responses relative to those of the control group. RESULTS: TB patients had a higher body temperature and pulse rate, as well as a platelet counts, ferritin, CRP and dopamine levels, with a tendency to higher cortisol levels compared to the control group. Conversely, they had a lower BMI, haemoglobin, leucocyte count, MCV and epinephrine levels than the control group. CONCLUSIONS: Patients with active pulmonary tuberculosis were documented to mount an acute stress response which was more intense than that of a control group of patients with surgical/medical conditions other than tuberculosis.
Assuntos
Proteínas de Fase Aguda/análise , Catecolaminas/sangue , Hidrocortisona/sangue , Estresse Fisiológico , Tuberculose Pulmonar/sangue , Adolescente , Adulto , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , África do Sul , Centros de Atenção Terciária , Tuberculose Pulmonar/diagnóstico , Adulto JovemRESUMO
The recent global resurgence of severe infections caused by the Group A streptococcus (GAS) pathogen, Streptococcus pyogenes, has focused attention on this microbial pathogen, which produces an array of virulence factors, such as the pore-forming toxin, streptolysin O (SOT). Importantly, the interactions of SOT with human neutrophils (PMN), are not well understood. The current study was designed to investigate the effects of pretreatment of isolated human PMN with purified SOT on several pro-inflammatory activities, including generation of reactive oxygen species (ROS), degranulation (elastase release), influx of extracellular calcium (Ca2+) and release of extracellular DNA (NETosis), using chemiluminescence, spectrophotometric and fluorimetric procedures, respectively. Exposure of PMN to SOT alone caused modest production of ROS and elastase release, while pretreatment with the toxin caused significant augmentation of chemoattractant (fMLP)-activated ROS generation and release of elastase by activated PMN. These effects of treatment of PMN with SOT were associated with both a marked and sustained elevation of cytosolic Ca2+concentrations and significant increases in the concentrations of extracellular DNA, indicative of NETosis. The current study has identified a potential role for SOT in augmenting the Ca2+-dependent pro-inflammatory interactions of PMN, which, if operative in a clinical setting, may contribute to hyper-activation of PMN and GAS-mediated tissue injury.
Assuntos
Armadilhas Extracelulares , Neutrófilos , Streptococcus pyogenes , Estreptolisinas , Humanos , Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Degranulação Celular/efeitos dos fármacos , Células Cultivadas , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Inflamação/imunologia , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/efeitos dos fármacos , Elastase Pancreática/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Estreptolisinas/metabolismoRESUMO
Although pro-inflammatory mechanisms have been implicated in the pathogenesis of manganese (Mn²âº)-related neurological and respiratory disorders, relatively little is known about the potential of this metal to interact pro-oxidatively with human phagocytes. The primary objective of the current study was to investigate the effects of Mn²âº as MnCl2 (0.5-100 µM) on the generation of the reactive oxygen species (ROS), superoxide, hydrogen peroxide (H2O2), and hypohalous acids by isolated human blood neutrophils and monocyte-derived macrophages following activation of these cells with the chemotactic tripeptide, FMLP (1 µM), or the phorbol ester, PMA (25 ng/mL). Generation of ROS was measured using the combination of oxygen consumption, lucigenin/luminol-enhanced chemiluminescence, spectrofluorimetric detection of oxidation of 2,7-dichlorodihydrofluorescein, radiometric assessment of myeloperoxidase (MPO)-mediated protein iodination, release of MPO by ELISA, and spectrophotometric measurement of nitrite formation. Treatment of activated neutrophils with either FMLP or PMA resulted in significantly decreased reactivity of superoxide in the setting of increased formation of H2O2 and MPO-mediated iodination, with no detectable effects on either oxygen consumption or MPO release. Similar effects of the metal with respect to superoxide reactivity and H2O2 formation were observed with activated macrophages, while generation of NO was unaffected. Taken together with the findings of experiments using cell-free ROS-generating systems, these observations are compatible with a mechanism whereby Mn²âº, by acting as a superoxide dismutase mimetic, increases the formation of H2O2 by activated phagocytes. If operative in vivo, this mechanism may contribute to the toxicity of Mn²âº.
Assuntos
Poluentes Ocupacionais do Ar/farmacologia , Peróxido de Hidrogênio/metabolismo , Macrófagos/efeitos dos fármacos , Manganês/farmacologia , Neutrófilos/efeitos dos fármacos , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Poluentes Ocupacionais do Ar/química , Poluentes Ocupacionais do Ar/toxicidade , Catálise , Células Cultivadas , Cloretos/química , Cloretos/farmacologia , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/farmacologia , Sequestradores de Radicais Livres/toxicidade , Humanos , Peróxido de Hidrogênio/química , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/enzimologia , Macrófagos/imunologia , Manganês/química , Manganês/toxicidade , Compostos de Manganês/química , Compostos de Manganês/farmacologia , Intoxicação por Manganês/imunologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/citologia , Neutrófilos/enzimologia , Neutrófilos/imunologia , Exposição Ocupacional/efeitos adversos , Concentração Osmolar , Oxidantes/química , Oxidantes/toxicidade , Peroxidase/metabolismo , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The research question addressed in the current study was: do formoterol (1 and 10 nM) and montelukast (2 µM) possess interactive inhibitory effects on activated human neutrophils, particularly in relation to alterations in cyclic AMP and cytosolic Ca²(+) fluxes? Isolated human blood neutrophils were activated with the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) (1 µM) in combination with cytochalasin B (CB; 3 µM). Fura-2-acetoxymethyl ester-based spectrofluorimetry, lucigenin-enhanced chemiluminescence, colorimetric and flow cytometric procedures were used to measure cytosolic Ca²(+) fluxes, production of superoxide, elastase release and beta-2 integrin (CR3) expression, respectively, while cyclic AMP and leukotriene (LT)B4 were assayed using competitive binding ELISA procedures. Activation of the cells with fMLP/CB resulted in abrupt and sustained increases in cytosolic Ca²(+), as well as release of elastase and production of superoxide and LTB4, and expression of CR3, all of which were attenuated by formoterol and montelukast individually, and especially by the combination of these agents. These anti-inflammatory effects of each agent, as well as the combination, were associated with significant increases in cyclic AMP. The findings of the current study may explain the efficacy of montelukast and formoterol when used in combination with inhaled corticosteroids in the treatment of severe asthma, possibly by controlling neutrophil-driven inflammation of the airways.
Assuntos
Acetatos/farmacologia , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Etanolaminas/farmacologia , Antagonistas de Leucotrienos/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Quinolinas/farmacologia , Adulto , Albuterol/farmacologia , Antígenos CD18/análise , Canais de Cálcio/efeitos dos fármacos , AMP Cíclico/análise , Ciclopropanos , Citocalasina B/farmacologia , Interações Medicamentosas , Fumarato de Formoterol , Humanos , Leucotrieno B4/análise , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Elastase Pancreática/análise , Elastase Pancreática/metabolismo , Rolipram/farmacologia , Sulfetos , Superóxidos/análiseRESUMO
OBJECTIVES: This study was designed to investigate the neutrophil-targeted anti-inflammatory potential of posaconazole (0.1-5 microM, equivalent to 0.7-3.9 mg/L) by measuring the effects of this agent on the release of leukotriene B(4) (LTB(4)) and store-operated uptake of Ca(2+) following stimulation of human neutrophils with platelet-activating factor (200 nM). METHODS: LTB(4) release and uptake of Ca(2+) by the cells were measured using an enzyme immunoassay and fura-2/AM-based spectrofluorimetric procedures, respectively. RESULTS: Treatment of neutrophils with posaconazole resulted in dose-related attenuation of PAF-activated release of LTB(4) and influx of Ca(2+), which attained statistical significance at 1 microM of the antimycotic. CONCLUSIONS: Although primarily an antimycotic, posaconazole possesses secondary anti-inflammatory activities, which may contribute to the therapeutic efficacy of this agent in patients with sepsis.
Assuntos
Anti-Inflamatórios/farmacologia , Cálcio/metabolismo , Fatores Imunológicos/farmacologia , Leucotrieno B4/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Triazóis/farmacologia , Adulto , Humanos , Imunoensaio/métodos , Neutrófilos/imunologia , Fator de Ativação de Plaquetas/imunologia , Espectrometria de Fluorescência/métodos , Adulto JovemRESUMO
OBJECTIVE AND DESIGN: To investigate the sensitizing effects of the cysteinyl leukotrienes (CysLTs) C(4) and D(4) on the proinflammatory responses of chemoattractant-activated human neutrophils in vitro. MATERIALS: Neutrophils were isolated from venous blood taken from healthy, adult, human volunteers. TREATMENT: Cells were exposed to LTC(4) and LTD(4) (50-300 nM) prior to activation with 1 microM of N-formyl-L-methionyl- L-leucyl-L-phenylalanine (fMLF). METHODS: A fura-2/AM-based spectrofluorimetric procedure, lucigenin-enhanced chemiluminescence (LECL), a colourimetric method and an ELISA procedure, were used to measure Ca(2+) mobilization, superoxide production, elastase and MMP-8 release respectively following activation of LTC(4)/ D(4)-primed neutrophils with fMLF. Superoxide generation was also measured in the presence and absence of the CysLT receptor 1 antagonist, montelukast (100 nM). RESULTS: Exposure of neutrophils to either LTC(4) or LTD(4) alone had modest effects on Ca(2+) mobilization, while superoxide generation and elastase release were unaffected. However, relative to the responses of neutrophils activated with fMLF in the absence of the CysLTs, pre-treatment of the cells with either LTC(4)or LTD(4) resulted in significant, augmentation of fMLF-activated elastase and MMP-8 release and superoxide generation, which was attenuated by montelukast. CONCLUSION: These previously undocumented sensitizing interactions of LTs C(4) and D(4) with neutrophils may contribute to the activation of these cells in acute and chronic inflammation of both atopic and non-atopic aetiology, while identifying a role for montelukast in regulating neutrophil reactivity.
Assuntos
Fatores Quimiotáticos/imunologia , Inflamação/imunologia , Leucotrieno C4/farmacologia , Leucotrieno D4/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Acetatos/metabolismo , Adulto , Ciclopropanos , Corantes Fluorescentes/metabolismo , Fura-2/análogos & derivados , Fura-2/metabolismo , Humanos , Antagonistas de Leucotrienos/metabolismo , Leucotrieno C4/imunologia , Leucotrieno D4/imunologia , Metaloproteinase 8 da Matriz/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Neutrófilos/citologia , Elastase Pancreática/metabolismo , Quinolinas/metabolismo , Sulfetos , Superóxidos/metabolismoRESUMO
Exposure of human neutrophils to ultraviolet radiation (UVR) in vitro was accompanied by activation of superoxide generation and preferential release of secondary granules. These pro-oxidative interactions of UVR with neutrophils were dependent on intact cellular membrane-associated oxidative metabolism and were mediated almost exclusively by the UVB component of UVR. Irradiation of neutrophils was also associated with release of arachidonic acid from membrane phospholipids, implicating involvement of phospholipase A2 (PLA2) in the pro-oxidative activity of UVR. The pro-oxidative interactions of UVR with neutrophils were mimicked by coincubation of the cells with reagent arachidonate or lysophosphatidylcholine (LPC), whereas the PLA2 inhibitor 4-p-bromophenacyl bromide, as well as the LPC- and arachidonate complex-forming agent alpha-tocopherol, inhibited these pro-oxidative interactions of UVR with phagocytes. Because phagocyte-derived reactive oxidants are cytotoxic, immunosuppressive, and carcinogenic, these agents are potential mediators of UVR-mediated tissue damage and tumorigenesis.
Assuntos
Neutrófilos/metabolismo , Neutrófilos/efeitos da radiação , Raios Ultravioleta , Ácido Araquidônico/farmacologia , Cálcio/metabolismo , Degranulação Celular/efeitos da radiação , Membrana Celular/metabolismo , Movimento Celular/efeitos da radiação , Humanos , Membranas Intracelulares/metabolismo , Lisofosfatidilcolinas/farmacologia , Neutrófilos/fisiologia , Oxirredução/efeitos da radiação , Fagocitose/efeitos da radiação , Superóxidos/metabolismoRESUMO
Relationships among the plasma levels of vitamin E (VE), the numbers and prooxidative activities of circulating phagocytes, serum alpha-1-protease inhibitor (API), and pulmonary functions were investigated in 83 asymptomatic male cigarette smokers and 65 nonsmoking controls. Plasma levels of VE, of cholesterol, and of API were measured using high performance liquid chromatography, spectrophotometry, and nephelometry, respectively, whereas reactive oxidant (ROS) generation by activated blood phagocytes was measured using a whole blood luciginen-enhanced chemiluminescence method. Smoking was associated with significantly increased circulating neutrophil counts (p 0.0001), serum API (p 0.0001) and phagocyte-derived ROS-generation (p 0.0001), and decreased spirometric values (FEV1: p 0.0138 and FEF25-75: p 0.0654). Plasma VE and cholesterol levels were not significantly different between smokers and nonsmokers. However, in smokers both plasma VE and cholesterol correlated significantly and positively with serum API (r 0.24, p 0.03 and r 0.30, p 0.005, respectively), neutrophil counts (r 0.24, p 0.03 and r 0.25, p 0.03, respectively), and phagocyte-derived ROS-generation (r 0.32, p 0.003 and r 0.32, p 0.003, respectively), and significantly and inversely with FEV1 (r -0.23, p 0.03 and r -0.22, p 0.04, respectively) and FEF25-75 (r -0.32, p 0.003 and r -0.26, p 0.02, respectively). In nonsmokers plasma VE, but not cholesterol, was positively correlated with FEV1 (r 0.34, p 0.007) and FEF25-75 (r 0.40, p 0.001). The results suggest that VE protects the lungs of both smokers and nonsmokers and may act as a mobilizable antioxidant in response to smoking-induced oxidative stress.
Assuntos
Pulmão/fisiologia , Pulmão/fisiopatologia , Estresse Oxidativo , Fagócitos/fisiologia , Fumar/fisiopatologia , Vitamina E/sangue , Adulto , Estudos de Casos e Controles , Colesterol/sangue , Humanos , Contagem de Leucócitos , Leucócitos/efeitos dos fármacos , Leucócitos/fisiologia , Masculino , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Valores de Referência , Testes de Função Respiratória , Fumar/sangue , Espirometria , alfa 1-Antitripsina/metabolismoRESUMO
We have investigated the effects of the adenosine A(2A) receptor agonist CGS 21680 (0.01 - 1 microM) on reactive oxidant production by, and elastase release from FMLP-activated human neutrophils, as well as on cytosolic Ca(2+) fluxes and intracellular concentrations of cyclic AMP. Oxidant production, elastase release and cyclic AMP were assayed using lucigenin-enhanced chemiluminescence, colourimetric and radioimmunoassay procedures respectively, while cytosolic Ca(2+) fluxes were measured by fura-2 spectrofluorimetry in combination with radiometric procedures which distinguish between net efflux and influx of the cation. Treatment of neutrophils with CGS 21680 did not affect the FMLP-activated release of Ca(2+) from intracellular stores, but resulted in dose-related acceleration of the rate of decline in fura-2 fluorescence, as well as decreases in both efflux and store-operated influx of Ca(2+), compatible with enhancement of resequestration of the cation by the endo-membrane Ca(2+)-ATPase. These effects on neutrophil Ca(2+) handling were associated with increased intracellular cyclic AMP and with inhibition of oxidant production and release of elastase. In contrast, treatment of neutrophils with the selective A(2A) receptor antagonist, ZM 241385 (2.5 microM), prevented the transient increase in cyclic AMP in FMLP-activated neutrophils which was associated with delayed sequestration of incoming Ca(2+) during store-operated influx. The CGS 21680-mediated reduction of Ca(2+) efflux from FMLP-activated neutrophils was also antagonized by pretreatment of the cells with ZM 241385 (2.5 microM), as well as by thapsigargin (1 microM), an inhibitor of the endo-membrane Ca(2+)-ATPase. ZM 241385 also neutralized the cyclic AMP-elevating and anti-inflammatory interactions of CGS 21680 with neutrophils. We conclude that A(2A) receptors regulate the pro-inflammatory activities of human neutrophils by promoting cyclic AMP-dependent sequestration of cytosolic Ca(2+).
Assuntos
Cálcio/metabolismo , Neutrófilos/efeitos dos fármacos , Adenosina/análogos & derivados , Adenosina/farmacologia , Radioisótopos de Cálcio , AMP Cíclico/metabolismo , Citosol/química , Citosol/metabolismo , Relação Dose-Resposta a Droga , Fura-2 , Humanos , Inflamação/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/citologia , Neutrófilos/metabolismo , Elastase Pancreática/efeitos dos fármacos , Elastase Pancreática/metabolismo , Fenetilaminas/farmacologia , Agonistas do Receptor Purinérgico P1 , Antagonistas do Receptor Purinérgico P2 , Superóxidos/metabolismo , Tapsigargina/farmacologia , Triazinas/farmacologia , Triazóis/farmacologiaRESUMO
1. We have investigated the effects of salmeterol (0.3-50 microM) on several pro-inflammatory activities of human neutrophils in vitro. 2. Oxidant production by FMLP- and calcium ionophore (A23187)-activated neutrophils was particularly sensitive to inhibition by low concentrations (0.3-3 microM) of salmeterol, while the responses of phorbol myristate acetate- and opsonised zymosan-stimulated cells were affected only by higher concentrations (3-50 microM) of the drug. At these concentrations salmeterol is not cytotoxic, nor does it act as a scavenger of superoxide. 3. These anti-oxidative interactions of salmeterol with neutrophils were insensitive to propranolol but could be eliminated by washing the cells, or by pretreatment with low concentrations (1-2 microM) of the pro-oxidative, membrane-destabilizing phospholipids, lysophosphatidylcholine (LPC), platelet activating factor (PAF) and lysoPAF (LPAF). 4. At concentrations of 6.25-50 microM salmeterol interfered with several other activities of stimulated neutrophils, including intracellular calcium fluxes, phospholipase A2 activity and synthesis of PAF. 5. In an assay of membrane-stabilizing activity, salmeterol (25 and 50 microM) neutralized the haemolytic action of LPC, PAF and LPAF. 6. Of the other commonly used beta 2-adrenoceptor agonists, fenoterol, and formoterol, but not salbutamol, caused moderate inhibition of neutrophil oxidant generation by a superoxide-scavenging mechanism. However, unlike salmeterol, these agents possessed only weak membrane stabilizing properties. 7. We conclude that salmeterol antagonizes the pro-inflammatory, pro-oxidative activity of several bioactive lipids implicated in the pathogenesis of bronchial asthma, by a mechanism related to the membrane-stabilizing, rather than to the beta 2-agonist properties of this agent.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Albuterol/análogos & derivados , Anti-Inflamatórios/farmacologia , Neutrófilos/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Adulto , Albuterol/farmacologia , Asma/fisiopatologia , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Etanolaminas/farmacologia , Fenoterol/farmacologia , Fumarato de Formoterol , Humanos , Técnicas In Vitro , NADH NADPH Oxirredutases/metabolismo , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Fosfolipases A/metabolismo , Fosfolipases A2 , Fator de Ativação de Plaquetas/biossíntese , Proteína Quinase C/metabolismo , Xinafoato de Salmeterol , Superóxidos/metabolismoRESUMO
1. We have investigated the effects of the selective phosphodiesterase (PDE) type 4 inhibitor, rolipram (0.01-1 microM) on cytosolic Ca2+ fluxes in FMLP-activated human neutrophils, as well as on superoxide production by, and release of elastase from, these cells. 2. Cytosolic Ca2+ fluxes were measured by use of fura-2 spectrofluorimetry in combination with a radiometric procedure that enables distinction between net efflux and influx of the cation. Superoxide production and elastase release were measured by lucigenin-enhanced chemiluminescence and a colorimetric procedure, respectively. 3. Pretreatment of neutrophils with rolipram did not affect the FMLP-activated release of Ca2+ from intracellular stores, but was associated with dose-related acceleration of the rate of decline in fura-2 fluorescence and with decreased efflux, as well as store-operated influx of 45Ca2+, indicative of enhancement of resequestration of the cation by the endo-membrane Ca2+-ATPase. 4. Inhibition of superoxide production and elastase release was observed at concentrations of rolipram which accelerated the clearance of Ca2+ from the cytosol of FMLP-activated neutrophils. 5. These effects of rolipram on FMLP-activated Ca2+ fluxes, superoxide generation and elastase release were mimicked by pretreatment of neutrophils with dibutyryl cyclic AMP (0.5-4 mM), while theophylline (10-150 microM), a non-specific PDE inhibitor, as well as the beta2-agonist, salbutamol, were less effective. 6. We conclude that rolipram deactivates FMLP-stimulated human neutrophils by enhancement of cyclic AMP-dependent resequestration of cytosolic Ca2+.
Assuntos
Bucladesina/farmacologia , Cálcio/metabolismo , Citosol/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Pirrolidinonas/farmacologia , Agonistas Adrenérgicos beta/farmacologia , Adulto , Albuterol/farmacologia , Radioisótopos de Cálcio , Corantes Fluorescentes , Fura-2 , Humanos , Técnicas In Vitro , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Rolipram , Superóxidos/metabolismo , Teofilina/farmacologiaRESUMO
We have investigated the effects of the beta-adrenoreceptor-blocking agent, propranolol (9-300 microM) on several pro-inflammatory activities of human neutrophils in vitro. Superoxide production by calcium ionophore (A23187)-activated neutrophils was particularly sensitive to inhibition by low concentrations (9-18.7 microM) of this drug. However, inhibition of superoxide generation by neutrophils activated with phorbol myristate acetate (PMA), opsonized zymosan (OZ), and arachidonate (AA) only occurred with higher concentrations of propranolol, and coincided with decreased intracellular calcium fluxes, phospholipase A2 (PLA2) activity and synthesis of platelet-activating factor (PAF). Propranolol possessed neither cytotoxic nor superoxide-scavenging properties but, using a haemolytic assay of membrane-stabilizing activity, this agent neutralized the membrane-disruptive effects of the bioactive phospholipids, lysophosphatidylcholine (LPC), PAF, and lysoPAF(LPAF). A mechanistic relationship between the anti-oxidative and membrane-stabilizing properties of propranolol was suggested by the observation that pretreatment of neutrophils with LPC or PAF eliminated the inhibitory effects of the drug on superoxide generation by PMA-activated neutrophils. Dexpropranolol, a stereoisomer with minimal beta-blocking activity, and propranolol were equally effective with respect to their membrane-stabilizing and anti-oxidative interactions with neutrophils, but several other beta-blocking agents (atenolol, metoprolol, sotalol, and timolol) did not possess these activities. Inhibition of oxidant generation is, therefore, not a common property of beta-blocking agents and, in the case of propranolol, appears to occur as a consequence of membrane-stabilization rather than by beta-receptor-directed effects.
Assuntos
Antioxidantes/farmacologia , Neutrófilos/efeitos dos fármacos , Propranolol/farmacologia , Acridinas , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Medições Luminescentes , Lisofosfatidilcolinas/metabolismo , Ativação de Neutrófilo , Neutrófilos/metabolismo , Fator de Ativação de Plaquetas/metabolismoRESUMO
This study was designed to evaluate the effects of epinephrine (0.01-1 microM) on superoxide production by, and release of elastase from human neutrophils activated with the chemotactic tripeptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) (1 microM) in vitro, and to relate alterations in these responses to changes in adenosine 3,5' cyclic monophosphate (cAMP) and cytosolic free Ca(2+). Cyclic AMP, superoxide production and elastase release were measured by radioimmunoassay, lucigenin-enhanced chemiluminescence, and a colorimetric procedure respectively. Cytosolic Ca(2+) fluxes were measured by fura-2 spectrofluorimetry in combination with radiometric procedures that enable distinction between net efflux and influx of the cation. Epinephrine treatment of neutrophils resulted in increased cAMP and dose-related inhibition of both superoxide production and elastase release, which was potentiated by the type 4 phosphodiesterase inhibitor, rolipram, and attenuated by propranolol, but not by selective beta(1)-, alpha(1)- or alpha(2)-adrenoreceptor antagonists. Although epinephrine did not affect the FMLP-activated abruptly-occurring increase in fura-2 fluorescence intensity, indicating no effects on the release of Ca(2+) from neutrophil intracellular stores, this agent accelerated the rate of decline in fluorescence in the setting of decreased efflux and a reduction in store-operated influx of Ca(2+). These effects of epinephrine on the clearance of Ca(2+) from the cytosol of FMLP-activated neutrophils were attenuated by propranolol, and are compatible with enhancement of the activity of the cAMP-dependent Ca(2+) sequestering/resequestering endo-membrane Ca(2+)-ATPase. We conclude that epinephrine down-regulates the pro-inflammatory activities of neutrophils by cAMP-mediated enhancement of the clearance of cytosolic Ca(2+).
Assuntos
Epinefrina/farmacologia , Neutrófilos/efeitos dos fármacos , Elastase Pancreática/metabolismo , Superóxidos/metabolismo , Adulto , Transporte Biológico/efeitos dos fármacos , Cálcio/metabolismo , Radioisótopos de Cálcio , AMP Cíclico/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Corantes Fluorescentes/metabolismo , Fura-2/metabolismo , Humanos , Técnicas In Vitro , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Vasoconstritores/farmacologiaRESUMO
This study was undertaken to identify the adenosine receptor (AR) subtypes which down-regulate the proinflammatory activities of human neutrophils, as well as the involvement of adenosine 3',5'-cyclic monophosphate (cAMP) and its relationship to cellular handling of Ca(2+) in mediating these effects. Neutrophils were treated with varying concentrations (0.01-1 microM) of AR agonists operative at A(1) (N(6)-cyclopentyladenosine, CPA), A(2A) (2(4-[(2-carboxyethyl)phenyl]ethylamino)-5'-N-ethylcarboxamidoadenosi ne, CGS 21680), and A(3) (N(6)-(3-iodobenzyl-5'-N-methylcarbamoyladenosine, IB-MECA) receptors, after which they were activated with the chemoattractant, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP, 1 microM). Intracellular cAMP, superoxide, and elastase were assayed using radioimmunoassay, lucigenin-enhanced chemiluminescence (LECL), and colorimetric procedures, respectively, while changes in the concentrations of cytosolic Ca(2+) were monitored by fura-2-based spectrofluorimetry. CGS 21680, at all concentrations tested, inhibited superoxide production in a dose-related manner, while CPA and IB-MECA were effective only at the highest concentrations tested (0.5-1 microM). The release of elastase from activated neutrophils was also inhibited by all three AR agonists, but was more sensitive to CGS 21680 and IB-MECA than was superoxide production. The inhibitory effects of all 3 agonists on superoxide production and elastase release were associated with accelerated clearance of Ca(2+) from the cytosol of activated neutrophils, and were effectively neutralized by pretreatment of the cells with the highly selective A(2A)R antagonist, ZM 241385 (4-(2-[7-amino-2-(2-furyl)[1, 2,4]triazolo[2,3-a][1,3,5]triazin-5yl amino]ethyl)phenol). Increased cAMP was detected in neutrophils treated with CGS 21680 and IB-MECA (1 microM). These data support the involvement of the A(2A)R subtype in the suppression of superoxide production and degranulation by activated human neutrophils, probably by cAMP-mediated alterations in Ca(2+) handling.
Assuntos
Adenosina/análogos & derivados , Adenosina/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Neutrófilos/efeitos dos fármacos , Fenetilaminas/farmacologia , Receptores Purinérgicos P1/metabolismo , Degranulação Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Fura-2/metabolismo , Humanos , Técnicas In Vitro , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Elastase Pancreática/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor A2A de Adenosina , Receptores Purinérgicos P1/efeitos dos fármacosRESUMO
SETTING: Inflammation-related oxidative stress has been implicated in the pathogenesis of lung fibrosis and dysfunction in patients with pulmonary tuberculosis. OBJECTIVE: To investigate the effects of antimicrobial chemotherapy and smoking status on the plasma concentrations of the anti-oxidative nutrients vitamin C, vitamin E and beta-carotene, as well as those of iron, lipid peroxides and the acute phase reactants C-reactive protein (CRP) and ferritin. DESIGN: A total of 41 patients with active pulmonary tuberculosis were studied at the outset and after 6 months of antimicrobial chemotherapy. RESULTS: Initial plasma concentrations of vitamin C and beta-carotene were low, returning to normal values after chemotherapy in the non-smokers, but not in the smokers, while those of vitamin E remained low throughout in both groups. Ferritin and CRP concentrations decreased significantly following chemotherapy, with the former higher in smokers than in non-smokers. Serum lipid peroxides were elevated in patients with pulmonary tuberculosis and were unaffected by chemotherapy or smoking habits, while iron levels were not significantly affected by chemotherapy. Although residual dysfunction and infiltration were evident, pulmonary function (FEV1) and radiographic score improved equally in both smokers and non-smokers following antimicrobial chemotherapy. CONCLUSIONS: Even after 6 months of apparently successful antimicrobial chemotherapy, pulmonary tuberculosis is associated with increased oxidative stress, which is unrelated to cigarette smoking and characterized by increased levels of circulating lipid peroxides and low concentrations of plasma vitamin E.
Assuntos
Antioxidantes/metabolismo , Estresse Oxidativo , Tuberculose Pulmonar/sangue , Tuberculose Pulmonar/tratamento farmacológico , Proteínas de Fase Aguda/análise , Adulto , Antituberculosos/uso terapêutico , Ácido Ascórbico/sangue , Feminino , Humanos , Ferro/sangue , Peróxidos Lipídicos/sangue , Masculino , Pessoa de Meia-Idade , Testes de Função Respiratória , Fumar/sangue , Fumar/fisiopatologia , Tuberculose Pulmonar/fisiopatologia , Vitamina E/sangue , beta Caroteno/sangueRESUMO
In this study we have investigated the effects of serum (10, 20, and 40% final concentrations) on the activity of NADPH-oxidase and energy metabolism of activated human neutrophils in vitro. Neutrophils were stimulated with FMLP, PMA, or opsonized zymosan in the presence and absence of serum, and generation of reactive oxidants by intact cells was measured using lucigenin-enhanced chemiluminescence (LECL). This method was also used to measure NADPH-oxidase activity in purified membrane preparations from neutrophils activated with PMA in the presence or absence of serum. Cellular ATP levels and activities of the various glycolytic enzymes were assayed using CL and spectrophotometric procedures, respectively. Inclusion of serum with neutrophils during exposure to the various stimuli of membrane-associated oxidative metabolism caused significant enhancement of the LECL responses of intact cells as well as of the activity of NADPH-oxidase in purified membranes prepared from PMA-activated neutrophils. In the absence of serum, the ATP levels and activity of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), but not the other glycolytic enzymes, were decreased in activated neutrophils, while inclusion of serum preserved neutrophil ATP levels and activity of G3PDH. Serum supplementation of the cell-suspending medium appears to promote optimum activity of NADPH-oxidase in stimulated neutrophils by preventing premature, oxidative inactivation of cellular energy metabolism.
Assuntos
Fenômenos Fisiológicos Sanguíneos , NADH NADPH Oxirredutases/metabolismo , Neutrófilos/enzimologia , Explosão Respiratória/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Adulto , Animais , Bovinos , Relação Dose-Resposta a Droga , Glicólise/efeitos dos fármacos , Humanos , Medições Luminescentes , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADPH Oxidases , Neutrófilos/efeitos dos fármacos , Proteínas Opsonizantes/farmacologia , Oxirredução , Inibidores da Síntese de Proteínas/farmacologia , Soroalbumina Bovina/farmacologia , Superóxido Dismutase/farmacologia , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Zimosan/farmacologiaRESUMO
The effects of the macrolide antimicrobial agents azithromycin, clarithromycin, erythromycin and roxithromycin on the prooxidative activity of stimulated human neutrophils have been investigated in vitro. Superoxide generation by activated neutrophils was measured by lucigenin-enhanced chemiluminescence. At the concentrations used (2.5-80 micrograms/ml) none of the test agents was cytotoxic, nor did they possess superoxide-scavenging properties. Treatment of neutrophils with all 4 macrolides was accompanied by dose-related inhibition of superoxide production by cells activated with FMLP or the calcium ionophore (A23187), while the responses activated by phorbol myristate acetate (PMA) or opsonized zymosan were minimally affected. The anti-oxidative interactions of roxithromycin with FMLP-activated neutrophils were neutralized by pretreatment of the cells with low, non-cytotoxic concentrations (0.5 microgram/ml) of the prooxidative, proinflammatory bioactive phospholipids, lysophosphatidylcholine (LPC), platelet-activating factor (PAF) and lyso-PAF (LPAF). Using an assay of membrane-stabilizing activity, the macrolides antagonized the membrane-disruptive effects of LPC, PAF and LPAF, without affecting enzymes involved in their synthesis. These membrane-stabilizing interactions of macrolides with neutrophils may counteract the proinflammatory, prooxidative activity of several bioactive lipids which have been implicated in the pathogenesis of bronchial asthma.
Assuntos
Antibacterianos/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Membrana Celular/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Adulto , Antiasmáticos/farmacologia , Asma/tratamento farmacológico , Asma/fisiopatologia , Azitromicina/farmacologia , Calcimicina/farmacologia , Membrana Celular/fisiologia , Claritromicina/farmacologia , Eritromicina/farmacologia , Humanos , Ionóforos/farmacologia , Medições Luminescentes , Lisofosfatidilcolinas/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/metabolismo , Neutrófilos/ultraestrutura , Fator de Ativação de Plaquetas/análogos & derivados , Fator de Ativação de Plaquetas/farmacologia , Explosão Respiratória/efeitos dos fármacos , Roxitromicina/farmacologia , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Zimosan/farmacologiaRESUMO
The effects were studied of the non-specific phosphodiesterase inhibitor, theophylline (37.5-300 microM), on intracellular levels of cyclic adenosine monophosphate (cAMP) and superoxide generation following exposure of human neutrophils to four different stimuli of neutrophil membrane-associated oxidative metabolism, each of which utilizes a different transductional mechanism to activate NADPH-oxidase, in vitro. Exposure of neutrophils to FMLP (1 microM), the calcium ionophore A23187 (1 microM), and opsonized zymosan (OZ, 0.5 mg/ml) was accompanied by activation of superoxide production and increased concentrations of intracellular cAMP. Inclusion of theophylline resulted in augmentation of cAMP and inhibition of superoxide production by these stimuli. These negative effects of theophylline on neutrophil superoxide generation were mimicked by dibutyryl cAMP and 8-bromo-cGMP, while the inhibitory activity of all 3 agents was antagonized by the protein kinase A inhibitor KT 5720, but not by the G-kinase inhibitor KT 5823. Unlike FMLP, OZ and A23187, intracellular cAMP levels did not increase in cells activated with phorbol-12-myristate-13-acetate (PMA, 25 ng/ml), while oxidant production activated by this stimulus was insensitive to the inhibitory effects of theophylline. These observations suggest that the beneficial, anti-inflammatory interactions of theophylline with human neutrophils are related to the phosphodiesterase inhibitory properties of this agent, and are selective for those pro-inflammatory stimuli which elevate cAMP, resulting in enhanced activity of protein kinase A and inhibition of the production of potentially harmful reactive oxidants by these cells.
Assuntos
Carbazóis , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Neutrófilos/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Superóxidos/metabolismo , Teofilina/farmacologia , Alcaloides/farmacologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Inibidores Enzimáticos , Humanos , Indóis/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Estresse Oxidativo , Pirróis/farmacologiaRESUMO
The effects of the bioactive phospholipids (PL), platelet-activating factor (PAF), lyso-PAF, and lysophosphatidylcholine (LPC) on the beat frequency and structural integrity of human ciliated respiratory epithelium were studied in vitro, in the presence or absence of polymorphonuclear leukocytes (PMNL), the antimicrobial agents, roxithromycin, clarithromycin, and azithromycin and the antioxidative enzymes catalase and superoxide dismutase (SOD). All three PL caused dose-dependent slowing of ciliary beat frequency (CBF) and epithelial damage (ED) at concentrations > or = 1 microgram/ml, which were unaffected by inclusion of the antimicrobial agents and antioxidative enzymes. When epithelial strips were exposed to the combination of PMNL and PL, there was significant potentiation of ciliary dysfunction and ED, which was ameliorated by pretreatment of the PMNL with the antimicrobial agents or by inclusion of catalase, but not SOD. These results demonstrate that LPC, PAF, and lyso-PAF cause epithelial damage by direct mechanisms which are oxidant-independent, as well as by indirect mechanisms involving phagocyte-derived reactive oxidants. Macrolides and azalide antimicrobial agents may have beneficial effects on airway inflammation in asthma and microbial infections by protecting ciliated epithelium against oxidative damage inflicted by PL-sensitized phagocytes.