Rooperol, an inhibitor of cytokine synthesis, decreases the respiratory burst in human and rat leukocytes and macrophages

Luminol-enhanced chemiluminescence was measured in fresh whole human blood, or human neutrophils isolated from heparinized blood, human alveolar macrophages and rat alveolar macrophages stimulated with bacterial endotoxin (LPS). Tetraacetate esters of rooperol, a dicatechol showing anticytokine activity, added to cells simultaneously with LPS inhibited the respiratory burst. The effective concentrations of rooperol were in the range of 1-10 μM depending on cell type and corresponded well with inhibition of nitric oxide production by rat alveolar macrophages. Thus rooperol may reduce some effects of excessive phagocytic activity and inflammatory reaction but by quenching free radicals production may also diminish the resistance to bacterial infections.


Introduction
Phagocytes from blood, i.e. polymorphonuclear leukocytes and monocytes, and tissue macrophages, are involved in complex defence mechanisms against pathogenic bacteria. During phagocytosis they exhibit increased metabolic activity known as respiratory burst which generates superoxide as its primary product. Superoxide is formed in effect of oxygen reduction by NADPH-oxidase, a multicomponent enzyme present in the cell membrane of phagocytes. 1 In a series of further reactions, superoxide gives rise to reactive oxygen intermediates (ROI) such as hydrogen peroxide, hydroxyl radical and hypochlorous acid which serve as microbicidal agents but produced in excess may damage host cells. 2 Respiratory burst is induced not only by intact bacteria but also by opsonized particles, endotoxin, some cytokines (tumour necrosis factor, TNFa, interferon-c, IFN-c), N-formylated chemoattractant peptides and activators of kinase C (phorbol esters). 1 Bactericidal activity is also generated by reactive nitrogen intermediates, such as nitric oxide (NO) 3 possessing a broad spectrum of biological activities and produced in macrophages by inducible nitric oxide synthase (iNOS). 4 The interaction between reactive oxygen and nitrogen intermediates leads to peroxynitrite and may increase cytotoxicity or in ammation. 3 Thus it is not surprising that simultaneous inhibition of nitric oxide synthase and NADPH oxidase suppress in ammatory arthritis. 5 Moreover, some antioxidants show anti-in ammatory properties and inhibit synthesis of acute phase cytokines. 6,7 We recently demonstrated that rooperol, a plant dicatechol, decreases production of tumour necrosis factor (TNFa), interleukin-1 (IL-1) and interleukin-6 (IL-6) in LPS-stimulated human and rat macrophages and NO production in rat macrophages. 7,8 Here we describe the effect of rooperol tetraacetate on the respiratory burst in human and rat phagocytes.

Cells, isolation and culture
Twenty ml of whole bood was taken from the nger of healthy volunteers (25± 48 years of age) and used immediately for respiratory burst measurement. Neutrophils were isolated from fresh heparinized human blood of healthy donors by centrifugation with Mono-Poly resolving medium, washed in Hanks' Balanced Salt Solution (HBSS) without Ca 2 and c. 5000 cells were used for experiments. Human lung macrophages were obtained by bre-optic bronchoscopy of patients examined for diagnostic purposes following the guidelines of the American Thoracic Society. 9 The cells were collected in sterile phosphate buffered saline (PBS), centrifuged, washed in PBS and c. 5000 cells were used for each experiment. The lungs of killed by ether overdose rats were lavaged with 3 3 10 ml of cold PBS. After centrifugation, the cell pellet was washed in cold PBS and 2 3 10 4 cells were used for respiratory burst estimation.
NO production by rat alveolar macrophages was evaluated in 24 h culture of the cells. Isolated rat macrophages were seeded on 12well plates (Costar) in RPMI with 8% fetal bovine serum and antibiotics. After 2 h the unattached cells were discarded, and the adherent cells were cultured for 24 h. Then rooperol tetraacetate (RTA) and/or LPS (500 ng/ml) in RPMI containing 2% FBS was added. NO was estimated in the medium collected after 24 h culturing the cells with the drugs.

Estimation of chemiluminescence (CL) and NO production
Whole blood in HBSS containing glucose and 0.5% FCS was incubated for 10 min with luminol (1.4 mM), RTA (0.6, 2, 5 and 10 mM) and/or 10 ng/ml LPS. The addition of 50 ml of latex beads (2 3 10 10 ) was the start point of chemiluminescence estimation in Scintillation Spectrometer Rackbeta 1211, LKB. 10 Neutrophils and macrophages were incubated with luminol, RTA and/or LPS for 20 min. As a result of exposure to latex beads respiratory burst occurs and its intensity depends on phagocytes priming by LPS.
NO production was estimated with Griess reagent and nitrite concentration was determined by using dilutions of sodium nitrite in water as a standard. 11 Rooperol tetraacetate at 20 mM concentration had no effect on nitrite assay.

Statistical analysis
Data were analysed by paired Student's t-test.

Results
Effect of rooperol on the respiratory burst of human cells Addition of bacterial endotoxin (10 ng/ml) to fresh human blood or isolated neutrophils followed by latex beads increased chemiluminescence within the rst 15± 20 min (Table 1) and a decline occurred usually around 30± 40 min after the addition of latex beads. However, considerable individual variations were observed, especially with full blood, in which the maximum was often delayed or prolonged (Fig. 1). When rooperol tetracetate was added to fresh human blood simultaneously with LPS a marked reduction of respiratory burst was found ( Fig. 1a and 1ax) and occasionally the chemiluminescence was reduced below control (Fig. 1ax). Similar inhibitory effects of rooperol were also visible with neutrophils isolated from human peripheral blood (Fig. 1b) and human alveolar macrophages (Fig. 1c). These effects were dose-dependent and for human neutrophils and macrophages almost total inhibition of respiratory burst occurred at 10 mM RTA ( Fig.  1b and 1c). In the case of full blood or isolated neutrophils 10 mM RTA reduced latex-beadsinduced chemiluminescence by approximately three-fold (Fig. 2). A similar inhibition of chemiluminescence was observed in one experiment with isolated human blood monocytes (data not shown). Table 1. Stimulatory effect of LPS (10 ng/ml) on latexbeads-induced chemiluminescence of cells present in 20 ml of fresh human blood or isolated neutrophils (, 5000 cells). The chemiluminescence was recorded after 3, 9, 15 and 21 min following the addition of latex beads. The results are reported as percentage of control preparation (without LPS) assumed as 100% at any given time in each experiment: mean SD of between three and eight independent experiments. Statistically signi® cant differences at P , 0.05, Effect of rooperol on the respiratory burst and NO production by rat macrophages The respiratory burst of rat alveolar macrophages primed with LPS was also quenched by rooperol (Fig. 3). In the conditions used, rat alveolar macrophages response to RTA was less pronounced in comparison with human macrophages. Moreover, the maximal response to 10 ng/ml of LPS occurred faster (in about 5 min after phagocytosis initiation) but also declined earlier (Fig. 3). When rat alveolar macrophages were cultured for 24 h with 500 ng/ml of LPS they produced signi cant amounts of nitric oxide determined as nitrite (123.8 17.8 nmol/ ml, n 6). Rooperol reduced NO production in a dose-dependent manner (Fig. 4), the IC 50 was found to be between 5 and 10 mM.

Discussion
Acetate esters of rooperol, a dicatechol from the South African plant Hypoxis rooperi, were shown to inhibit production of TNFa, IL-1b and IL-6 in endotoxin-stimulated human alveolar macrophages, blood monocytes and U937 promonocytic cell line. 7 Rooperol tetraacetate (RTA) was effective in low micromolar range (IC 50 10± 20 mM) and at this concentration had no effect on cell viability as shown by the incorporation of 14 C-leucine into macro- FIG. 1. The inhibitory effect of RTA (0.6, 2, 5 and 10 mM) on chemiluminescence induced by LPS (10 ng/ml) and latex beads suspension (50 ml) in white cells present in 20 ml of full human blood (a and ax); in 5000 neutrophils isolated from peripheral human blood (b); or in 5000 human alveolar macrophages (c). Fresh full blood was preincubated with luminol, RTA and/or LPS for 10 min, isolated neutrophils and macrophages for 20 min, and then latex beads were added and chemiluminescence measured.
Mediators of In¯ammation´Vol 6´1997 5 5 phage proteins and cellular contents of lactate dehydrogenase. Subsequent studies revealed that RTA may inhibit cytokine synthesis in promonocytic U937 cell line at the pretranslational level. 8 All those results suggested that rooperol esters are potential anti-in ammatory drugs.
The experiments described here clearly demonstrate another anti-in ammatory activity of rooperol, i.e. inhibition of the respiratory burst. We found that RTA at micromolar concentrations signi cantly decreased chemiluminescence of human blood leukocytes and alveolar macrophages, as well as of rat alveolar macrophages, that had been primed with LPS and stimulated to phagocytosis by latex beads. The mechanism of respiratory burst inhibition by rooperol is not elucidated but may involve events related to LPS priming, 12 direct interaction with enzymatic pathway of oxygen radicals formed by NADP oxidase, 1 or involvement of cell membrane constitutents including protein kinase C as described for coenzyme Q 13 which is structurally related to rooperol.
Our results con rm earlier observations that RTA can also inhibit NO production in rat alveolar macrophages 7 and murine endothelial cells. 14 The broad spectrum of biological activities of rooperol suggests that this compound may be bene cial in the treatment of various in ammatory diseases, such as asthma or rheumatoid arthritis, in which overproduction of acute phase cytokines, nitric oxide and reactive oxygen intermediates has been reported. 5,15 It should be remembered, however, that by quenching free radicals production rooperol may also diminish the resistance of organism to bacterial infections. 16 and neutrophils (5000 cells) (striped bar), 9 min after latex addition. Full blood was preincubated for 10 min, neutrophils for 20 min with luminol, RTA and/or LPS. Chemiluminescence caused by LPS alone was assumed as 100%. Mean ( SD) of three to six experiments. Statistically signi® cant differences at P , 0.05, P , 0.02 and P , 0.01.