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1.
Free Radic Biol Med ; 115: 421-435, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29248721

RESUMO

In this study, we report the ability of a set of eight 3-phenylcoumarin derivatives bearing 6,7- or 5,7-dihydroxyl groups, free or acetylated, bound to the benzopyrone moiety, to modulate the effector functions of human neutrophils. In general, (i) 6,7-disubstituted compounds (5, 6, 19, 20) downmodulated the Fcγ receptor-mediated neutrophil oxidative metabolism more strongly than 5,7-disubstituted compounds (21, 22, 23, 24), and (ii) hydroxylated compounds (5, 19, 21, 23) downmodulated this neutrophil function more effectively than their acetylated counterparts (6, 20, 22, 24, respectively). Compounds 5 (6,7-dihydroxy-3-[3',4'-methylenedioxyphenyl]-coumarin) and 19 (6,7-dihydroxy-3-[3',4'-dihydroxyphenyl]-coumarin) effectively downmodulated the neutrophil oxidative metabolism elicited via Fcγ and/or complement receptors. Compound 5 also downmodulated the immune complex-stimulated phagocytosis, degranulation of elastase, and production and release of neutrophil extracellular traps, as well as the human neutrophil chemotaxis towards n-formyl-methionyl-leucyl-phenylalanine, without altering the expression level of formyl peptide receptor type 1. Both compounds 5 and 19 did not impair the neutrophil capacity to recognize and kill Candida albicans. Docking calculations revealed that compounds 5 and 19 directly interacted with three catalytic residues - Gln-91, His-95, and Arg-239 - inside the myeloperoxidase active site. Together, these findings indicate that (i) inhibition of reactive oxygen species generation and degranulation of elastase are closely associated with downmodulation of release of neutrophil extracellular traps; and (ii) compound 5 can be a prototype for the development of novel immunomodulating drugs to treat immune complex-mediated inflammatory diseases.


Assuntos
Anti-Inflamatórios/farmacologia , Cumarínicos/farmacologia , Armadilhas Extracelulares/metabolismo , Neutrófilos/fisiologia , Elastase Pancreática/metabolismo , Receptores de Complemento/metabolismo , Receptores de IgG/metabolismo , Anti-Inflamatórios/química , Células Cultivadas , Cumarínicos/química , Humanos , Imunomodulação , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/metabolismo , Fagocitose , Espécies Reativas de Oxigênio/metabolismo
2.
J Pharm Pharmacol ; 69(12): 1829-1845, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28994118

RESUMO

OBJECTIVES: To examine whether the hydroalcoholic extract from Baccharis dracunculifolia leaves (BdE) modulates the human neutrophil oxidative metabolism, degranulation, phagocytosis and microbial killing capacity. METHODS: In-vitro assays based on chemiluminescence, spectrophotometry, flow cytometry and polarimetry were used, as well as docking calculations. KEY FINDINGS: At concentrations that effectively suppressed the neutrophil oxidative metabolism elicited by soluble and particulate stimuli (<10 µg/ml), without clear signs of cytotoxicity, BdE (1) inhibited NADPH oxidase and myeloperoxidase activity; (2) scavenged H2 O2 and HOCl; (3) weakly inhibited phagocytosis; and (4) did not affect neutrophil degranulation and microbial killing capacity, the expression levels of TLR2, TLR4, FcγRIIa, FcγRIIIb and CR3 and the activity of elastase and lysozyme. Caffeic acid, one of the major B. dracunculifolia secondary metabolites, did not inhibit phagocytosis but interfered in the myeloperoxidase-H2 O2 -HOCl system by scavenging H2 O2 and HOCl, and interacting with the catalytic residues His-95, Arg-239 and Gln-91. CONCLUSIONS: BdE selectively modulates the effector functions of human neutrophils, inhibits the activity of key enzymes and scavenges physiological oxidant species. Caffeic acid contributes to lower the levels of oxidant species. Our findings help to unravel the mechanisms by which these natural products exert immunomodulatory action towards neutrophils.


Assuntos
Baccharis/química , Fatores Imunológicos/farmacologia , Neutrófilos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Adulto , Ácidos Cafeicos/isolamento & purificação , Ácidos Cafeicos/farmacologia , Citometria de Fluxo , Sequestradores de Radicais Livres/isolamento & purificação , Sequestradores de Radicais Livres/farmacologia , Humanos , Fatores Imunológicos/isolamento & purificação , Luminescência , Neutrófilos/metabolismo , Fagocitose/efeitos dos fármacos , Folhas de Planta , Espectrofotometria
3.
Artigo em Inglês | MEDLINE | ID: mdl-25450839

RESUMO

INTRODUCTION: This study aims to optimize some experimental conditions of a flow cytometric assay to examine the human neutrophil ability to phagocytose immune complexes (ICs) via Fcγ and complement receptors (FcγR and CR, respectively). The parameters assessed were: number of cells, concentration of ICs, reaction time, pH and concentration of the Trypan Blue quenching solution. METHODS: Neutrophils were isolated from peripheral blood of healthy volunteers. Precipitated ICs composed of IgG and fluorescein isothiocyanate (FITC)-labeled ovalbumin, opsonized or not with serum complement, were used to trigger the neutrophil phagocytosis via FcγR, CR, and FcγR+CR. Fluorescence of the internalized ICs was measured by flow cytometry, after quenching the extracellular fluorescence with Trypan Blue. RESULTS: The optimal experimental conditions established for the phagocytosis assay were: 1 × 10(6) cells mL(-1) and 40 µg mL(-1) FITC-labeled ICs, incubated for 30 min, at 37°C, in 0.5 mL of reaction volume. Trypan Blue solution at 1.25 mg mL(-1) pH4.4 was the best fluorescence quencher of FITC-labeled ICs attached to the outer surface of neutrophils. DISCUSSION: The selected experimental conditions were viable to evaluate IC phagocytosis by neutrophils; they are also suitable to compare the efficiency of IC phagocytosis mediated by FcγR and CR classes of membrane receptors, alone or in combination. This method finds application in studies of (i) the receptor-specific phagocytic function of normal and pathogenic neutrophils as well as (ii) the impact of drugs and therapies on this essential effector function of neutrophils.


Assuntos
Complexo Antígeno-Anticorpo/fisiologia , Neutrófilos/fisiologia , Fagocitose/fisiologia , Receptores de Complemento/fisiologia , Receptores de IgG/fisiologia , Células Cultivadas , Citometria de Fluxo , Humanos , Espécies Reativas de Oxigênio
4.
Z Naturforsch C J Biosci ; 69(7-8): 346-56, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25265855

RESUMO

Stimulated human neutrophils exhibit increased net oxygen consumption (NOC) due to the conversion of O2 into the superoxide anion by the NADPH oxidase enzymatic complex during the respiratory burst. In several inflammatory diseases, overproduction of these oxidants causes tissue damage. The present study aims to: (a) optimize the experimental conditions used to measure the NOC in serum-opsonized zymosan (OZ)- and insoluble immune complex (i-IC)-stimulated human and rabbit neutrophils; and (b) compare the effect of four flavonols (quercetin, myricetin, kaempferol, and galangin) on this activity. We used a Clark-type oxygen electrode to measure the NOC of stimulated neutrophils. Eliciting the neutrophil respiratory burst with OZ and i-IC yielded similar maximum O2 uptake levels within the same species, but the human neutrophil NOC was almost four times higher than the rabbit neutrophil NOC. The optimal experimental conditions established for both cell types were 4 x 10(6) neutrophils mL(-1), 2 mg mL(-1) OZ, and 240 microg mL(-1) i-IC. Upon stimulation with OZ or i-IC, the tested flavonols reduced the human and rabbit neutrophil NOC in the same order of potency--quercetin and galangin were the most and the least potent, respectively. These compounds were around four times more effective in inhibiting the rabbit as compared to the human neutrophil NOC, respectively. The four flavonols were not toxic to human or rabbit neutrophils. The experimental conditions used are suitable for both the determination of human and rabbit neutrophil NOC and for the assessment of the modulatory effects of natural compounds on these activities. The relationship between the level of NOC and the inhibitory potency of the flavonols suggests that rabbit neutrophils can be useful experimental models to predict the effect of drugs on immune complex-stimulated human neutrophils.


Assuntos
Complexo Antígeno-Anticorpo/imunologia , Flavonóis/farmacologia , Neutrófilos/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Animais , Humanos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Coelhos
5.
Int Immunopharmacol ; 21(1): 102-11, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24797916

RESUMO

Rheumatoid arthritis (RA) patients usually exhibit immune complex (IC) deposition and increased neutrophil activation in the joint. In this study, we assessed how four flavonols (galangin, kaempferol, quercetin, and myricetin) modulate the effector functions of healthy individuals' and active RA patients' IC-stimulated neutrophils. We measured superoxide anion and total reactive oxygen species production using lucigenin (CL-luc)- and luminol (CL-lum)-enhanced chemiluminescence assays, respectively. Galangin, kaempferol, and quercetin inhibited CL-lum to the same degree (mean IC50=2.5 µM). At 2.5 µM, quercetin and galangin suppressed nearly 65% CL-lum of active RA patients' neutrophils. Quercetin inhibited CL-luc the most effectively (IC50=1.71±0.36 µM). The four flavonols diminished myeloperoxidase activity, but they did not decrease NADPH oxidase activity, phagocytosis, microbial killing, or cell viability of neutrophils. The ability of the flavonols to scavenge hypochlorous acid and chloramines, but not H2O2, depended on the hydroxylation degree of the flavonol B-ring. Therefore, at physiologically relevant concentrations, the flavonols partially inhibited the oxidative metabolism of IC-stimulated neutrophils without affecting the other investigated effector functions. Using these compounds to modulate IC-mediated neutrophil activation is a promising safe therapeutic strategy to control inflammation in active RA patients.


Assuntos
Anti-Inflamatórios/farmacologia , Artrite Reumatoide/tratamento farmacológico , Flavonoides/farmacologia , Quempferóis/farmacologia , Neutrófilos/efeitos dos fármacos , Quercetina/farmacologia , Adulto , Anti-Inflamatórios/química , Complexo Antígeno-Anticorpo/imunologia , Degranulação Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Flavonoides/química , Humanos , Quempferóis/química , Pessoa de Meia-Idade , Neutrófilos/imunologia , Oxirredução/efeitos dos fármacos , Peroxidase/metabolismo , Quercetina/química , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade
6.
Chem Biol Interact ; 206(1): 63-75, 2013 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-23994743

RESUMO

In the present study, we assessed whether 7-hydroxycoumarin (umbelliferone), 7-hydroxy-4-methylcoumarin, and their acetylated analogs modulate some of the effector functions of human neutrophils and display antioxidant activity. These compounds decreased the ability of neutrophils to generate superoxide anion, release primary granule enzymes, and kill Candida albicans. Cytotoxicity did not mediate their inhibitory effect, at least under the assessed conditions. These coumarins scavenged hypochlorous acid and protected ascorbic acid from electrochemical oxidation in cell-free systems. On the other hand, the four coumarins increased the luminol-enhanced chemiluminescence of human neutrophils stimulated with phorbol-12-myristate-13-acetate and serum-opsonized zymosan. Oxidation of the hydroxylated coumarins by the neutrophil myeloperoxidase produced highly reactive coumarin radical intermediates, which mediated the prooxidant effect observed in the luminol-enhanced chemiluminescence assay. These species also oxidized ascorbic acid and the spin traps α-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 5-dimethyl-1-pyrroline-N-oxide. Therefore, 7-hydroxycoumarin and the derivatives investigated here were able to modulate the effector functions of human neutrophils and scavenge reactive oxidizing species; they also generated reactive coumarin derivatives in the presence of myeloperoxidase. Acetylation of the free hydroxyl group, but not addition of the 4-methyl group, suppressed the biological effects of 7-hydroxycoumarin. These findings help clarify how 7-hydroxycoumarin acts on neutrophils to produce relevant anti-inflammatory effects.


Assuntos
Antifúngicos/farmacologia , Antioxidantes/farmacologia , Candida albicans/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Umbeliferonas/farmacologia , Ânions/antagonistas & inibidores , Ânions/metabolismo , Antifúngicos/química , Antioxidantes/química , Relação Dose-Resposta a Droga , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade , Umbeliferonas/química
7.
Int Immunopharmacol ; 15(2): 387-94, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23333455

RESUMO

Immune complex (IC) deposition in tissues triggers the release of harmful oxidant and lytic compounds by neutrophils. We examined how ten 3-phenylcoumarin derivatives affect the reactive oxygen species (ROS) production by IC-stimulated human neutrophils. Most of the 3-phenylcoumarins inhibited the luminol-enhanced chemiluminescence (CL-lum) more strongly than they inhibited the lucigenin-enhanced chemiluminescence (CL-luc), without clear signs of toxicity. The most effective CL-lum inhibitors, 6,7-dihydroxy-3-[3',4'-methylenedioxyphenyl]-coumarin (5) and 6,7-dihydroxy-3-[3',4'-dihydroxyphenyl]-coumarin (19), also inhibited myeloperoxidase activity more potently and had higher hypochlorous acid scavenging ability, but did not affect the NADPH-oxidase activity. The type, number, and position of the substituent influenced the pharmacological effects of 3-phenylcoumarins; however, the structural requirements for CL-lum and CL-luc inhibition were a little different. Compounds 5 and 19 are promising prototypes of therapeutic molecules to modulate ROS production by neutrophils in IC-mediated inflammatory diseases.


Assuntos
Cumarínicos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Neutrófilos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Complexo Antígeno-Anticorpo/imunologia , Células Cultivadas , Cumarínicos/química , Descoberta de Drogas , Sequestradores de Radicais Livres/química , Humanos , Doenças do Complexo Imune/imunologia , Imunomodulação , Medições Luminescentes , Terapia de Alvo Molecular , Neutrófilos/imunologia , Oxirredução/efeitos dos fármacos , Peroxidase/metabolismo , Relação Estrutura-Atividade
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