- Research article
- Open Access
- Open Peer Review
Anvillea garcinii extract inhibits the oxidative burst of primary human neutrophils
BMC Complementary and Alternative Medicine volume 16, Article number: 433 (2016)
Anvillea garcinii Coss. & Durieu (Anv) plant is used as a traditional North African medicine against several diseases associated with inflammation. At inflammatory sites, reactive oxygen species (ROS) produced in excess by activated phagocyte NADPH oxidase (NOX2) can accentuate inflammatory responses. Thus, we investigated if Anv-water soluble polysaccharides could modulate primary human neutrophil oxidative burst in vitro.
Human neutrophils were isolated from fresh whole blood and O2.- generation was measured by cytochrome c reduction assays. Western blots were used to analyse the translocation of PKC, p47phox (a key component of NOX2 activity) to neutrophil plasma membrane. Also, myeloperoxidase (MPO) release in the extracellular medium was studied by western blots. Flow cytometric analysis was used to detect CD11b membrane expression.
Water soluble polysaccharides from Anv dose-dependently inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLF)- and phorbol myristate acetate (PMA)-induced O2.- generation by human neutrophils. Moreover, Anv-polysaccharides strongly inhibited PMA-induced PKCβ and p47phox translocation to membranes and p47phox phosphorylation on Ser328, a main PKC target. In contrast, polysaccharides extract from Zygophyllum gaetulum plant, which is also used as a traditional North African medicine against inflammatory diseases, was ineffective on this PKCβ-p47phox pathway. Further, Anv inhibited important neutrophil degranulation markers corresponding to myeloperoxidase (MPO) release and CD11b membrane expression.
The process of down-regulating NADPH oxidase by polysaccharides extracts from Anv provides new insights into the mechanism of Anv’s anti-inflammatory actions.
Increased reactive oxygen species (ROS) can damage healthy bystander tissues, thereby contributing to several inflammatory diseases such as rheumatoid arthritis and inflammatory bowel diseases . Neutrophil is the main ROS producing phagocyte which play an essential role in host defence against microbial pathogens and in inflammation. In response to stimulating agents such as the bacterial peptide N-formyl-methionyl-leucylphenyl-alanine (fMLF), neutrophils release large amounts of superoxide anion (O2.-) and other reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), used by myeloperoxidase (MPO) to produce hypochloric acid, in a phenomenon called the respiratory burst . Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase or NOX2, the enzyme responsible for superoxide anion production by phagocytes, is a multicomponent enzyme system consisting of membrane associated cytochrome b558 (composed of gp91phox and p22phox) and the cytosolic components p47phox, p67phox, p40phox, and rac1/2 (a small GTPase) [3, 4]. Neutrophil activation leads to phosphorylation of the cytosolic components such as p47phox, a protein which plays an important role in the assembly and activation of NADPH oxidase.
Activated neutrophils and several associated products such as ROS, myeloperoxidase (MPO), elastase and inflammatory adipokines have been found in synovial fluid of patients with rheumatoid polyarthritis [5, 6]. Neutrophils are highly mobile cells which are densely packed with secretory granules. Degranulation, a mechanism independent of ROS production, can also be incriminated as being a main cause of pulmonary disorders such as severe asphyxic episodes of asthma . The dual role of neutrophils has been further highlighted during respiratory viral infection whereby it contributed to beneficial antiviral responses as well as detrimental tissue pathology and cellular inflammation . Thus, the inhibition of excessive oxidant production and degranulation represent an important therapeutic target and could in part explain the beneficial effects of certain natural products used against inflammatory disorders. To this end, bioactive products isolated from medicinal plant such as Garcinia buchananii and Ficus aurantiaca Griff have been shown to be antioxidative by respectively displaying in vitro H2O2 scavenging activity  or inhibiting neutrophil ROS production . Indeed, integration of the wealthy African medication for several diseases including inflammation in western medicine is considered as a promising challenge . Medicinal plant extracts are usually made with water and water-soluble polysaccharides isolated from plants have attracted much attention because of their broad spectrum of therapeutic properties and relative low toxicity .
In this context, we investigated if water-soluble polysaccharide-extract from an endemic plant which grows in northern Africa and particularly in Morocco and Algeria, Anv, could have anti-inflammatory effects. This plant is known by traditional healers to possess several medicinal properties in North-African countries . For example, their crude extracts as well as their infusions are widely used to treat colds, diabetes, stomach and liver diseases often associated with inflammation and they are also antiseptic and antispasmodic [14–16].
Our study shows that Anvillea garcinii Coss. & Durieu water-soluble polysaccharide extract was efficient in inhibiting ROS production and degranulation of primary human phagocytes. Indeed, Anv was efficient in inhibiting neutrophil superoxide anion induced by PMA or fMLF. Analysis of the mechanism indicated a selective inhibition of a strong PKC phosphorylation site, Ser 328 on p47phox crucial for the assembly and function of neutrophil NOX2.
Phorbol myristate acetate (PMA), formyl-methionyl-leucylphenylalanine (fMLF), Tween 20, luminol, cytochrome c, horse radish peroxidase (HRP), phosphatases inhibitors and proteases inhibitors, ATP, HBSS, acetylsalicylic acid and PBS and other chemicals were purchased from Sigma Aldrich (Saint-Quentin Fallavier, France). Dextran T500 and Ficoll were purchased from GE Healthcare (Orsay, France). HEPES was from life technologies (Saint Aubin, France). SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis), and Western blot were purchased from Bio-Rad (Richmond, CA, USA). Antibodies against protein kinase C were from GeneTex (Irvine, CA, USA). Antibodies against, p22, βactin as well as secondary HRP-labeled goat anti-rabbit antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany). The anti-p47phox antibody was kindly provided by Dr. B. M. Babior (The Scripps Research Institute, La Jolla, CA, USA). The use of our rabbit polyclonal antibodies against phospho-sites p47phox (phospho-Ser328) have been described previously .
Anvillea garcinii collection and preparation of water-soluble polysaccharide extract
Anvillea garcinii Coss. & Durieu (Anv) was collected from Biskra Ghardaya (north of Algerian Sahara) and was taxonomically authenticated by Dr. Ali Zitouni (University 8 Mai 1945 of Guelma, Algeria) and deposited at the herbarium of the university with the voucher specimen; (No.A.g.2010-1). A standardized water-soluble polysaccharide extract was prepared as described previously . Briefly, 170 g of Anv powder, corresponding to the plant aerial part, was suspended in distilled water (1 g/20 mL) and stirred for 3 h at 100 °C before cooling overnight at 8 °C. The supernatant obtained by centrifugation for 30 min at 4000 g was reduced to half volume by evaporation. Polysaccharides were precipitated from the filtrates by the adding four volumes of ethanol (95 %). The precipitates (16.48 g = 9.7 %) were collected by centrifugation for 30 min at 15,000 g and washed with ethanol and dried. The sample was dissolved in distilled water (1 g/50 mL), dialysed against distilled water for 72 h at 4 °C. The dialysis residue was treated with tri-chloro-acetic acid and sodium acetate to remove proteins. The crude polysaccharide mixtures were precipitated by ethanol and air dried. The percentage yield relative to dry samples weight was 3.18 % and the presence of proteins was not detected by colorimetric methods [19, 20]. Similarly, we also extracted water-soluble polysaccharides (2.48 % yield) from another identified traditional medicinal plant from the same region, Zygophyllum gaetulum Emb. & Maire (Zyg), . It was collected and identified by by Dr. Ali Zitouni and deposited at the above herbarium with the voucher (No.A.g.2010-2).
Neutrophil isolation from peripheral human blood
Freshly drawn blood from healthy subjects was used to isolate neutrophils by dextran sedimentation and Ficoll fractionation . After centrifugation at 400 g for 30 min, neutrophil and monocyte/lymphocyte rings were collected. Erythrocytes were removed from neutrophils by hypotonic lysis. Following isolation, neutrophils were suspended in appropriate medium, such as Hank’s balanced salt solution (HBSS). Cell purity and counting were determined by flow cytometry analysis (FACS Canto II cytometer (BD, San Jose, CA, USA) and Turks’ blue staining respectively. The viability of neutrophils with or without the polysaccharides was also determined by differential permeability of DNA-binding dyes using the ViaCount reagent by Guava easy Cyte (IFAS-Cytometry Bioscience, Millipore, St Quentin en Yvelines, France).
Measurement of superoxide production
To measure superoxide anion or O2.- production, isolated neutrophils cells (5 × 105) were suspended in 500 μL of HBSS containing 1 mg/mL cytochrome c in the presence or absence of different concentrations of Anv. The samples were placed in the thermostated chamber of a spectrophotometer (Uvikon, Thermo Fisher, Villebon, France) preheated at 37 °C. After 15 min, cells were stimulated with 10−6 M of N-formyl-methionyl-leucyl-phenylalanine (fMLF) or 100 ng/mL of phorbol myristate acetate (PMA). Changes in absorbance were measured at 550 nm for 10 min.
Evaluating Anv scavenging activity of O2.-
To evaluate O2.- scavenging activity, the O2.- generation substrate xanthine (200 μM) and cytochrome c (1 mg/mL) were incubated with different concentrations of polysaccharides of Anv at 30 °C during 15 min. The enzyme xanthine oxidase was added and the amount O2.- was measured by assaying the cytochrome c reduction at 550 nm in a spectrophotometer (Uvikon) for 10 min.
Effect of Anv on neutrophil degranulation
Neutrophils (5 × 106 per 500 μL HBSS) were incubated in the presence or the absence of different concentrations of Anv for 15 min at 37 °C. Then, they were stimulated with either 10−6 M fMLF for 5 min or 100 ng/mL PMA for 15 min. Degranulation was stopped by cooling cell aliquots in ice-cold methanol (−80 °C) for 5 s. Cells were then centrifuged (4 °C) for 8 min at 400 g. Supernatants were removed and denatured with Laemmli sample buffer  for western blot analysis.
Flow cytometric analysis of the CD11b expression at neutrophil surface
Isolated neutrophils (2.5 × 106 per 500 μL PBS) were incubated at 37 °C for 15 min in the presence or the absence of Anv extracts. Samples were then stimulated with fMLF (10−6 M) for 5 min. The reaction was stopped by cooling the cell aliquots on ice. 100 μL of each sample was then stained with 20 μL of PE-conjugated anti-human CD11b monoclonal antibody (BD Biosciences, San Jose, CA) for 15 min at room temperature in the dark. Then the cells were washed twice with PBS at room temperature and analyzed by flow cytometry using FACS Canto II cytometer (BD Biosciences, San Jose, CA, USA) as described .
PKCβ and p47phox translocation to neutrophils plasma membrane
The experiment was performed as described before , briefly neutrophils (50 × 106 per mL) in HBSS buffer were incubated for 15 min at 37 °C in the absence (control) or the presence of either Anv or Zyg extract at 300 ug/mL and then cells were stimulated with 100 ng/mL PMA for 8 min. The reaction was stopped by adding 4 volumes of ice-cold PBS and cells were pelleted by centrifugation at 400 g for 8 min at 4 °C. The suspended cells were disrupted by sonication and centrifuged at 400 g for 8 min. The postnuclear supernatant was loaded onto a discontinuous sucrose gradient (35 % sucrose, 15 % sucrose), centrifuged for 45 min at 150,000 g. The membrane fraction in the 15 % sucrose layer was collected and washed in PBS before denaturing in Laemmli sample buffer. The samples were boiled for 3 min and stored at -80 °C until use.
Western blot analysis
The different samples were subjected to 10 % SDS-PAGE using standard techniques . The separated proteins were transferred to nitrocellulose, which was blocked with 5 % non-fat dry milk in Tris-buffered saline with 0.1 % tween-20 (TBST) for 1 h. After blocking, the nitrocellulose membranes were incubated for 1 h at 37 °C or overnight at -4 °C with specific antibodies (Anti MPO (1:5000), anti-phosphoSer328-p47phox (1:2000), anti-p47phox (1:5000), anti-PKCβ or α (1:1000)) followed by three washes with TBST, the membranes were incubated with HRP-labelled goat anti-rabbit or anti-mousse antibody (1:5000). After three washes with TBST, revelation was performed by a chemiluminescence method (luminol reagent, Santa Cruz Biotechnology, Santa Cruz, CA), according to the manufacturer instructions and using Amersham Imager 600 (LifeSciences, Velizy, France). The intensity of bands was quantified by densitometry using the Image J analysis program (National Institute of Health, USA). Alternatively, the membranes were incubated with an alkaline phosphatase-conjugated goat anti-mouse or goat anti-rabbit and proteins were revealed with the NBT/BCIP reagents (Saint Louis, Missouri, USA) in the carbonate buffer (0.1 mM NaHCO3, 1 mM MgCl2, pH 9.8).
All results are expressed as mean ± SEM. One-way analysis of variance with the Tukey-Kramer post hoc test for multiple comparisons was implemented using GraphPad Prism version 6.0 for Windows (GraphPad Software, San Diego, CA).
Anv inhibited superoxide production by neutrophil in response to PMA
The effect of Anv on ROS production by human neutrophils was compared with that of Zyg, another North African medicinal plant Zyg, which is similarly used to treat against inflammatory diseases, even though they are of different genus. We also studied the established anti-inflammatory drug, aspirin  and fMLF or PMA was used to stimulate ROS production which was measured by using luminol-amplified chemiluminescence as shown in Additional file 1. Anv was a strong inhibitor of ROS production with a similar IC50 value (60 μg/mL) for either fMLF or PMA response while Zyg was poorly effective. The aspirin IC50 values were 200 μg/mL and 300 μg/mL for inhibiting fMLF or PMA response respectively. To gain further insight in the mechanism of Anv inhibition, we explore its effect on the production of superoxide anion, the first product of NADPH oxidase. We used the cytochrome c reduction assay which is a specific technique for measuring superoxide anion. As shown in (Fig. 1a) Anv dose-dependently inhibited O2.- production by human neutrophils stimulated with fMLF. Further, a dramatic inhibition of O2.- production was observed in response to PMA. We investigated if this inhibition was due to a scavenging of O2.- by Anv extract by using the in vitro xanthine oxidase production of O2.- . Results in (Fig. 1b), show that the production of O2.- was not significantly altered in the presence of increasing concentrations of Anv. Taken together, the results are in favour of an Anv effect on NADPH oxidase activity as opposed to a scavenging activity.
Anv inhibited p47phox and PKC translocation to the neutrophil plasma membrane in response to PMA
We investigated if Anv can modulate PMA-induced translocation of p47phox and PKC in neutrophils which represents a potential immuno-modulatory pathway. We compared the effect of Anv with the water-soluble polysaccharide extract of Zyg which did not modulate O2.- production by human neutrophils in response to PMA, thus suggesting the involvement of different mechanisms and represented a potential control versus active Anv (data not shown). The results in (Fig. 2a, left panel) show that neither Anv nor Zyg was able to promote the translocation of p47phox to neutrophil plasma membrane in the resting state. When neutrophils were incubated with PMA, it stimulated a strong translocation of p47phox. This PMA-induced translocation was significantly inhibited in the presence of Anv and not Zyg as indicated by the histogram. Similar results were obtained with PKCβ showing that Anv was effective in inhibiting PKCβ translocation to plasma membrane in response to PMA (Fig. 2b). Further, activation of NADPH oxidase in neutrophils by PMA is known to be accompanied by a strong phosphorylation of Ser328 in the regulatory subunit, p47phox . We detected little or no phosphorylation using a specific anti-phosphoSer328 antibody, in control samples (Fig. 2c), while PMA induced a strong Ser328 phosphorylation. Interestingly, Anv at the same concentration used above (300 μg/ml) significantly inhibited this phosphorylation of p47phox on Ser328 stimulated by PMA while Zyg was ineffective.
Anv inhibited neutrophil degranulation
We investigated whether Anv extract could affect other important neutrophil functions such as degranulation. To this end, neutrophils were incubated without (control) and with 76 to 300 μg/mL of Anv extracts and then stimulated with fMLF or PMA as described in methods. Then myeloperoxidase (MPO), the most abundant pro-inflammatory enzyme stored in azurophilic granules of neutrophils, was detected in the extracellular medium by using western blot analysis. The results (Fig. 3a and b) show that Anv was effective in inhibiting MPO release in the extracellular medium induced by either fMLF or PMA. Further, we analysed the effect of Anv on fMLF-induced degranulation of specific granules by measuring the neutrophil surface expression of CD11b. In (Fig. 3c), the data of flow cytometric analysis are presented as a percentage of control values (fMLF only) and showed that Anv inhibited CD11b expression at the neutrophil outer membrane. Taken together, the results suggest that Anv inhibits neutrophil degranulation processes.
Innate immunity serves as an essential first-line of defense against microbial pathogens and foreign substances. Phagocytic cells such as neutrophils play a key role in innate immunity because of their ability to recognize, ingest and destroy pathogens by oxidative and nonoxidative mechanisms. However, neutrophil hyper-activation has been shown to induce tissue injury and inflammatory reactions [1, 8]. Thus, inhibition of neutrophil hyper activation represents an interesting strategy to develop novel anti-inflammatory agents. There is increasing research in identifying novel natural plant products to integrate in conventional immunomodulators as they often have less side effects [11, 12]. Our data showed that water-soluble polysaccharides extract isolated from medicinal plants, Anv inhibited fMLF- and PMA-induced superoxide anion (O2.-) production in human neutrophils. Anv inhibited PMA-induced PKC activation resulting in the inhibition of PMA-induced phosphorylation and translocation of the NADPH oxidase subunit p47phox. Also, the Anv extract inhibited MPO degranulation. These results suggest that polysaccharides isolated from Anv could exert a strong anti-inflammatory effect by inhibiting neutrophil functions and by limiting reactive oxygen species (ROS) propagation to nearby tissues.
An important difference in the inhibitory effect of Anv was observed on O2.- produced in response to either fMLF and PMA. The pronounced effect on PMA versus fMLF may be due to different mechanisms including O2.- scavenging since ROS-scavenging activity has been observed with some polysaccharide extracts [25, 26]. Polysaccharide isolated from Artemisa tripartite has been shown to scavenge both H2O2 and O2.- and their capacity to scavenge may be due to difference in polysaccharide composition and size . The potent scavenging property of exogenous polysaccharides compounds represents a therapeutic potential for oxidative stress-associated inflammatory diseases. This has been recently highlighted in vivo by endogenous scavenging lectin such as Reg3 alpha which suppresses extracellular reactive oxygen species to protect from liver toxicity . In our study, we found that O2.- was not scavenged by Anv polysaccharide using an in vitro assay whereby superoxide anion (O2.-) is produced by xanthine oxidase .
We thus explored how Anv could modulate neutrophil extracellular production of O2.- by NADPH oxidase (NOX2) since several polysaccharide extracts have been shown to modulate phagocyte ROS production without or with scavenging activity [29, 30]. Interestingly, it was shown that while water-insoluble chitosan derivatives can activate neutrophils by a phagocytosis mechanism, soluble ones would prime neutrophils and potentiate oxidative burst . Thus, soluble polysaccharide extracts can have both stimulatory and inhibitory effects on neutrophil functions. In our study, it is unlikely that water-soluble polysaccharide extract of Anv directly inhibited the NOX2 enzyme since there was a preferential inhibition of O2.- production by NOX2 induced by two different stimulators, PMA or fMLF. Moreover, PMA represents not only a positive control but it is an appropriate tool to explore the signal transduction pathways involved in the activation of NOX2 and degranulation process of neutrophils [24, 31, 32]. PMA can directly activate the phospholipid-dependent protein kinase C, which translocate to the plasma membrane and by phospohorylating NADPH oxidase components such as p47phox participates in the assembly and activation of NOX2 . All PKC isoforms present in human neutrophils, including PKCβ are known to activate NADPH oxidase . The biological relevance of PKC pathway was sustained by the use of opsonised zymosan, components of yeast cell wall, which activated a PKC-dependent activation of NOX2 in neutrophils . We found that Anv inhibited the membrane translocation of p47phox and PKCβ induced by PMA. Water-soluble polysaccharide extract of another North African medicinal plant Zyg, which is similarly used as Anv against inflammatory diseases, was also studied in order to explore their mechanisms. Further, Zyg is sometimes given together with Anv as traditional medicine. In contrast to Anv, we found that Zyg barely inhibited O2.- production by neutrophils in response to either PMA or fMLF (data not shown). In line with this observation, we found that Zyg was ineffective in modulating the membrane translocation of p47phox and PKCβ induced by PMA. The production of superoxide anions by NOX2 in human neutrophil is accompanied by extensive phosphorylation of p47phox which is an excellent substrate for PKC . To further validate the action of Anv on PKC/ p47phox, we studied its effect on serine 328 of p47phox, which is one of the most phosphorylated serine residues by PKC. The results obtained by using our raised antibody against this phosphorylated residue showed that Anv polysaccharide was able to inhibit PMA-induced phosphorylation of serine 328 of p47phox as well as its translocation to neutrophil plasma membrane and Zyg extract was ineffective. Altogether, the results indicate a clear effect of water-soluble Anv polysaccharide extract on the modulation of PMA-induced superoxide anions.
The progression of inflammation is significantly affected by myeloperoxidase (MPO) released from azurophilic granules of neutrophils accumulated at inflammatory sites . MPO-dependent oxidative system can damage host tissue through the generation of oxidants. There are some data suggesting that polysaccharides may influence the traffic of different granules in neutrophils and their degranulation in phagosomes or the extracellular environment. It has been recently shown that water soluble polysaccharides extracted from Bupleurum chinense inhibited fMLF-induced HL60 cell recruitment and involved the inhibition of rac-1 activation . The traffic of cytosolic rac-1 or rac-2 to plasma membrane is crucial in the assembly of functional NADPH oxidase and may represent a site of inhibitory action for polysaccharide extracts. Moreover, rac-2 serves has a selective role in the degranulation of neutrophils, a process which is independent of ROS production . Further, the anti-inflammatory aqueous extract of Punia granatum has been shown to selectively inhibit MPO activity of human neutrophils which depends on degranulation process . In contrast, it has been shown that acidic polysaccharides isolated from the medicinal plant Tanacetum vulgare L. can enhance neutrophil MPO release . Thus, it is important to test the effect of Anv on neutrophil degranulation initiated by fMLF or PMA. The effect on azurophilic or specific granules was estimated by following MPO and CD11b expression respectively. We could show that Anv extract was more effective in inhibiting fMLF- than PMA-induced MPO release in the extracellular medium. Also, the surface expression of CD11b, a sensitive marker of PMN activation by fMLF was inhibited by Anv. Our results indicate that Anv polysaccharide can inhibit both PMA and fMLF induced degranulation processes which involve a large group of intracellular signalling molecules .
We demonstrated that Anv polysaccharides have a strong antioxidant action on neutrophils as summarized in Fig. 4. Moreover, Anv was very efficient at limiting NADPH oxidase activation by inhibiting the phosphorylation of p47phox and its translocation to the plasma membrane. In addition, the extract inhibited MPO degranulation. In conclusion, by inhibiting both extracellular ROS production and neutrophil’s degranulation, Anv has strong anti-inflammatory properties which make it a promising candidate for further medicinal applications.
- Anv :
Anvillea garcinii Coss. & Durieu
Phagocyte NADPH oxidase
Phorbol myristate acetate
- Zyg :
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We thank Dr. Tarek Bousetta and Dr. Stuart Moore from INSERM U1149, Paris France for their excellent scientific comments.
There were no grants.
Availability of data and materials
Specimens of the two tested plants were deposited at the herbarium of the university with the voucher specimen; (No.A.g.2010-1) for Anvillea garcinii Coss. & Durieu and voucher; (No.A.g.2010-2) for Zygophyllum gaetulum. Materials and data of our study are available to other researches upon request.
HB and DB collected the plants and prepared the extracts. HB and MH-N and VM conducted the experiments and the analysis and interpretation of data. J El-B, J-C M and DB conceived the research idea and prepared the final manuscript. All authors read and approved the paper before submission.
The authors declare that they have no competing interests.
Consent for publication
This information is not applicable.
Ethics approval and consent to participate
Blood was from healthy subjects who volunteered and came from the blood bank (EFS, Paris, France). This study was approved by our institutional Human Ethics Committee with reference number 2015012778 and there were written informed consents.
Dose-effect of Anvillea garcinii (Anv), Zygophyllum gaetulum (Zyg) and aspirin (Asp) on human neutrophils ROS production. Human neutrophils were incubated with increasing concentration of Anv (38–300 μg/mL), Zyg (38–300 μg/mL) or Asp (62–500 μg/mL) 15 min before stimulation with A) fMLF (10−6 M) or B) PMA (100ng/ mL). ROS was measured by luminol-amplified chemiluminescence and data are expressed as percentage to control (fLMF or PMA alone). All results are means ± SEM of three or more separate experiments. (DOC 35 kb)