Coumarinic derivatives show anti-inflammatory effects on alveolar macrophages, but their anti-elastase activity is essential to reduce lung inflammation in vivo

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Abstract

We have previously demonstrated the potency of coumarinic derivatives to inhibit human leukocyte elastase. Given the anti-inflammatory activities of some coumarins, we investigated the capacity of our coumarinic derivatives to inhibit inflammation and whether their anti-elastase activity was essential for their anti-inflammatory functions. All compounds studied were coumarinic derivatives displaying differential anti-proteinase activity. Coumarinic derivatives 1, 2, and 3 efficiently inhibited human leukocyte elastase in vitro, whereas the coumarinic derivative 4 did not show inhibitory activity. The anti-inflammatory effect of these compounds and a coumarin control, scopoletin, on interleukin-6 (IL-6), tumor necrosis factor (TNF), and macrophage chemotactic protein-1 (MCP-1) release was studied using lipopolysaccharide (LPS)-stimulated alveolar macrophages. The in vivo effect of compound 2, that inhibits elastase, and compound 4, that does not show proteinase inhibition, was investigated using a mouse model of LPS-induced lung inflammation and elastase-induced acute lung injury. All investigated coumarinic derivatives, regardless of their anti-proteinase activity, significantly inhibited IL-6 and TNF production by LPS-stimulated alveolar macrophages. However, only compounds 2, 3, and 4 significantly reduced MCP-1 release. Compound 2 attenuated LPS-induced leukocyte recruitment in bronchoalveolar lavage, whereas no inhibition was observed with compound 4 devoid of elastase inhibitory capacity. Interestingly, MCP-1 level was reduced in bronchoalveolar lavage of compound 4 treated mice, whereas TNF and IL-6 levels were not modulated by coumarins. Furthermore, compound 2, but not 4, reduced elastase induced lung injury. Our data suggest that although coumarinic derivatives have anti-inflammatory properties, their anti-elastase activity is essential to reduce lung inflammation in vivo.

Introduction

Neutrophil elastase is a serine proteinase with anti-microbial [1] and anti-fungal activities [2]. However, it is its proteolysis activity causing tissue destruction that is best known [3]. This potent serine proteinase is capable of digesting a panoply of matrix proteins [4] and is involved in numerous inflammatory respiratory diseases including emphysema [5], cystic fibrosis [6], chronic obstructive pulmonary disease [7], pulmonary fibrosis [8], and asthma [9], [10]. Given the biological effects of neutrophil elastase, there is a strong interest in developing potent and selective synthetic elastase inhibitors with low molecular weight and high bioavailability [11], [12]. Among the numerous elastase inhibitors developed, coumarinic derivatives have been shown to be promising molecules [13], [14].

Coumarins, consisting of fused benzene and α-pyrone rings, represent a vast family of compounds naturally found in plants [15]. These compounds show anti-viral and anti-bacterial activities [16] in addition to various anti-inflammatory abilities [13]. Several coumarins possess antioxidant capacity scavenging superoxide anion radicals [17], [18], reduce edema in rat paw carrageenan test and other inflammatory rodent models [19], and inhibit both the lipoxygenase and cyclooxygenase pathways of arachidonic acid metabolism [20], [21]. In murine macrophages, examples of coumarinic derivatives have been shown to inhibit lipopolysaccharide (LPS)-stimulated release of nitric oxide, interleukin (IL)-1β, IL-6, prostaglandin E2, and tumor necrosis factor (TNF) via the suppression of NF-кB activation [22], [23], [24].

In previous studies, we have modulated the coumarinic template to improve their anti-proteinase activity [25]. We demonstrated that several synthetic coumarinic derivatives are efficient low-molecular-weight, non peptidic and versatile proteinase inhibitors [26]. Aryl esters of 6-chloromethyl-2-oxo-2H-1-benzopyran-3-carboxylic acid behave as suicide substrates of α-chymotrypsin when a latent alkylating function at the 6-position (chloromethyl group) is present [26]. In contrast, pyridyl esters of the same acid and related structures displaying various substituents at the 6-position demonstrate that, in the absence of such an alkylating function, human leukocyte elastase is specifically inhibited through the formation of a transient acyl-enzyme [14], [25]. This inhibition is temporary as the enzyme slowly recovers its activity. Although the potency of these coumarinic derivatives against neutrophil elastase was demonstrated, their anti-inflammatory capacities have not been explored.

In the present study, we investigated the effect of coumarinic derivatives on LPS-stimulated alveolar macrophage mediator release and in an animal model of LPS-induced lung inflammation and elastase-induced acute lung injury. We screened four coumarinic derivatives (Table 1), three with neutrophil elastase inhibitory activity (pyridyl compounds 1, 2, and 3) and one without (phenylic compound 4). Compound 1 displays no substituent at the 6-position conversely to compound 3 (nitro group) and compounds 2 and 4 (ester group CH2OCOR). The anti-elastase activity was not necessary to reduce mediator release by LPS-stimulated alveolar macrophages, but was essential to inhibit lung inflammation in vivo.

Section snippets

Coumarinic derivatives

Compounds 14 were synthesized as previously described [14], [25], [27]. They have been dissolved in DMSO (ACS spectrophotometer grade, Sigma-Aldrich Chimie S.a.r.l., Lyon, France) before their use in biological experiments.

Animals

Female C57BL/6 mice weighing 12–15 g were obtained from CERJ (Le Genest-St-Isle, France). They had free access to food and water. All animal protocols were approved by the Animal Ethics Committee (CREEA) of Jacques Monod Institute.

Alveolar macrophages

NR8383 is a rat alveolar macrophage cell

Modulation of alveolar macrophage mediator release by coumarinic derivatives

To investigate the anti-inflammatory activities of coumarinic derivatives, alveolar macrophages were stimulated with LPS for 20 h in the presence of 1% DMSO (sham treated cells) or different concentrations of the coumarinic derivatives 1 to 4 or scopoletin, a coumarinic control. Pro-inflammatory mediator levels were measured in cell-free supernatants. Coumarinic derivatives significantly inhibited alveolar macrophage IL-6 release in a concentration-dependent manner (Fig. 1A). Coumarinic

Discussion

Alveolar macrophages are found throughout the respiratory tract and represent the most abundant immune cells in the airway lumen and alveolar space. These cells play a key role in the maintenance of the immunological homeostasis in the lung [35]. They are capable of inducing and inhibiting inflammatory responses thereby preventing lung injury. Coumarins have been shown to modulate macrophage functions [22], [23]. Given the functional difference between macrophages and alveolar macrophages [36],

Acknowledgments

This work was funded by CNRS, Université de Paris VI-Pierre et Marie Curie, and Foundation Alphonse L'Espérance (Laval Hospital). G. Tremblay was an invited professor at Université de Paris VI.

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