7-Hydroxycoumarin modulates the oxidative metabolism, degranulation and microbial killing of human neutrophils

Abstract

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.

Keywords: 1,1-diphenyl-2-picrylhydrazyl radical; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; 4-POBN; 4-aminobenzoic acid hydrazide; 5,5-dimethyl-1-pyrrolidone-2-oxyl-1; 5-dimethyl-1-pyrroline-N-oxide; 7-Hydroxycoumarin; AAPV; ABAH; Asc; CL; CL-luc; CL-lum; Candida albicans; DMPO; DMPOX; DMSO; DPPH; Degranulation; EPR; HBSS; HBSS-gel; HRP; Hank’s balanced saline solution; Hank’s balanced saline solution supplemented with gelatin; IC(50); LDH; MPO; MTT; Myeloperoxidase; N-succinyl-Ala-Ala-Val-p-nitroanilide; NADPH; Neutrophil; PI; PKC; PMA; Prooxidant; ROS; SAAVNA; SOD; SOZ; ascorbic acid; chemiluminescence; concentration inhibiting a biological response by 50%; dimethyl sulfoxide; electron paramagnetic resonance; horseradish peroxidase; lactate dehydrogenase; lucigenin-enhanced chemiluminescence; luminol-enhanced chemiluminescence; methoxy-succinyl-Ala-Ala-Pro-Val-chloromethylketone; myeloperoxidase; n-fMLP; n-formyl-methionyl-leucyl-phenylalanine; phorbol-12-myristate-13-acetate; propidium iodide; protein kinase C; reactive oxygen species; reduced form of nicotinamide adenine dinucleotide phosphate; serum-opsonized zymosan; superoxide dismutase; α-(4-pyridyl-1-oxide)-N-tert-butylnitrone.