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

Highlights

• 7-Hydroxycoumarin derivatives modulate the oxidative metabolism of human neutrophils.

• 7-Hydroxycoumarin impairs degranulation of neutrophils and killing of Candida albicans.

• Myeloperoxidase oxidizes 7-hydroxycoumarin to highly reactive free radicals.

• In the absence of MPO, the 7-hydroxycoumarin derivatives display antioxidant effect.

• 7-Hydroxycoumarin derivatives inhibit superoxide anion generation by 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.

Introduction

Neutrophils are professional killers of invading microorganisms and also contribute to the regulation of inflammatory processes. These cells secrete types of chemokines, cytokines, lipid mediators, granule proteins, and reactive oxygen species (ROS) that serve as signaling molecules for subsequent recruitment of inflammatory cells. These molecules help regulate the initiation of specific T and B cell immunity and the resolution phase of inflammation [1], [2], [3]. Thus, modulating the effector functions of neutrophils is a promising therapeutic strategy to manage inflammation.

Coumarin and its derivatives have arisen as promising natural products with potent anti-inflammatory effect and low toxicity to humans [4], [5], [6], [7], [8]. They constitute a large class of phenolic compounds produced by plants of different botanical families, primarily in Angiospermae [9]. The coumarin (1,2-benzopyrone; 5,6-benzo-α-pyrone) has been clinically used to treat cancer, lymphoedema, venous insufficiency and chronic infections [5], [6], [7], [8]. Coumarin alone is ineffective in treating some diseases, but it can increase the beneficial effects of other compounds in combination therapy [8]. However, it has a short half-life in humans due to hepatic metabolism (70–90%) to 7-hydroxycoumarin (Fig. 1) and its glucuronide. Coumarin is considered a prodrug and 7-hydroxycoumarin is the bioactive molecule [6], [8].

7-Hydroxycoumarin, also known as umbelliferone, occurs in plants and edible fruits and roots, such as golden apple, bitter orange, carrot, coriander, garden angelica, and mouse-ear hawkweed [10], [11]. This coumarin displays a wide range of beneficial bioactivities: it reduces lymphoedema in humans [6] and exerts anti-hyperglycemic [12], bronchodilating [10], antinociceptive [13], and antiedematogenic [4], [14], [15] effects in rats. The in vitro and in vivo antioxidant, anti-inflammatory and immunomodulatory effects of 7-hydroxycoumarin have been recently reported [4], [5], [11], [16], [17], [18], [19], [20], [21], [22]. This compound inhibits migration of neutrophils and eosinophils [10], degranulation of mast cells [15], release of NO by macrophages [19], [20], [21], release of cytokines [10], [13], biosynthesis of prostaglandin, and activity of cycloxygenase-2 and 5-lipoxygenase [4], [5], [14]. In addition, 7-hydroxycoumarin activates the protective immune responses of mice against influenza virus [19] and Salmonella typhimurium[22].

However, there are few reports on the action of 7-hydroxycoumarin on neutrophils. 7-Hydroxycoumarin and 7-hydroxy-4-methylcoumarin inhibit the generation of $O_2^{•-}$ by zymosan-stimulated rabbit neutrophils [23], phorbol-12-myristate-13-acetate (PMA)-stimulated human neutrophils, and n-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated rat leukocytes [5]. 7-Hydroxycoumarin also inhibits the migration of neutrophils in mouse models of inflammation [10], [13]. Therefore, the high potential of 7-hydroxycoumarin for the development of an effective anti-inflammatory drug and the fundamental contribution of neutrophils to the regulation of inflammatory processes have motivated us to evaluate the modulator effect of 7-hydroxycoumarin, 7-hydroxy-4-methylcoumarin, and their acetylated analogs (Fig. 1) on some effector functions of human neutrophils. In particular, we investigated their effects on the oxidative metabolism, degranulation, microbial killing ability, and myeloperoxidase and elastase activity of neutrophils. We also evaluated their physicochemical properties, antioxidant activity, and HOCl scavenging potential in cell-free systems.