Modulation of rat macrophage function by the Mangifera indica L. extracts Vimang and mangiferin

International Immunopharmacology 2 (2002) 797 – 806 www.elsevier.com/locate/intimp

Modulation of rat macrophage function by the Mangifera indica L. extracts Vimang and mangiferin D. Garcı´a a, R. Delgado b, F.M. Ubeira c, J. Leiro c,* a Departamento de Farmacia, Universidad Central de ‘‘Las Villas’’, Villa Clara, Cuba Laboratorio de Farmacologı´a, Centro de Quı´mica Farmace´utica, PO Box 16042, Havana, Cuba c Departamento de Microbiologı´a y Parasitologı´a, Laboratorio de Parasitologı´a, Facultad de Farmacia, Universidad de Santiago de Compostela, Santiago de Compostela 15782, Spain b

Received 23 October 2001; received in revised form 27 January 2002; accepted 11 February 2002

Abstract Vimang is an aqueous extract of Mangiferia indica L., traditionally used in Cuba as an anti-inflammatory, analgesic and antioxidant. In the present study, we investigated the effects of Vimang and of mangiferin (a C-glucosylxanthone present in the extract) on rat macrophage functions including phagocytic activity and the respiratory burst. Both Vimang and mangiferin showed inhibitory effects on macrophage activity: (a) intraperitoneal doses of only 50 – 250 mg/kg markedly reduced the number of macrophages in peritoneal exudate following intraperitoneal injection of thioglycollate 5 days previously (though there was no significant effect on the proportion of macrophages in the peritoneal-exudate cell population); (b) in vitro concentrations of 0.1 – 100 Ag/ml reduced the phagocytosis of yeasts cells by resident peritoneal and thioglycollate-elicited macrophages; (c) in vitro concentrations of 1 – 50 Ag/ml reduced nitric oxide (NO) production by thioglycollate-elicited macrophages stimulated in vitro with lipopolysaccharide (LPS) and IFNg; and (d) in vitro concentrations of 1 – 50 Ag/ml reduced the extracellular production of reactive oxygen species (ROS) by resident and thioglycollate-elicited macrophages stimulated in vitro with phorbol myristate acetate (PMA). These results suggest that components of Vimang, including the polyphenol mangiferin, have deppressor effects on the phagocytic and ROS production activities of rat macrophages and, thus, that they may be of value in the treatment of diseases of immunopathological origin characterized by the hyperactivation of phagocytic cells such as certain autoimmune disorders. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Mangifera indica L.; Mangiferin; Phagocytosis; Nitric oxide; Respiratory burst

1. Introduction Macrophages play an important role in innate and acquired immunity [1]. They are the first cells to participate in the immune response, and can be acti-

*

Corresponding author. Tel./fax: +34-81-593316. E-mail address: [email protected] (J. Leiro).

vated by a variety of stimuli. Their principal functions include the phagocytosis of foreign particles and the production of cytokines and reactive oxygen species (ROS) and nitrogen species (RNS) involved in the destruction of pathogens [2]. Although RNS and ROS are involved in host defense, overproduction of these species may contribute to the pathogenesis of inflammatory diseases [3,4]. Phagocytic cells, especially macrophages, have been implicated in immunopa-

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thological disorders related to oxidative stress including inflammatory diseases as such rheumatoid arthritis [5], myocardial reperfusion injury [6], hepatocellular oxidative DNA injury [7], and ulcerative colitis [8]. Vimang is an aqueous extract of Mangifera indica L., used traditionally in Cuba for its antiinflammatory, analgesic, and antioxidant properties [9– 11]. Several authors have reported other pharmacological activities of extracts of M. indica L., including spasmolytic, antiamoebic, antimicrobial, and antipyretic effects [12 –15]. These extracts are comprised of polyphenols, triterpenes, flavonoids, phytosterols, and microelements [16]. The major compound is mangiferin, a C-glucosylxanthone with antiviral, antitumor, antidiabetic, and antioxidant activity [17 –22]. In the present study, we evaluated the in vivo and in vitro effects of Vimang and mangiferin on macrophage function, specifically phagocytic activity, chemotaxis in response to inflammatory stimuli, and the respiratory burst (production of ROS and RNS).

2.2. Plant material, preparation of extracts, and treatments M. indica L. bark was collected from a mango orchard located in the region of Pinar del Rio, Cuba. A crude extract was prepared by decoction with a polar solvent for 1 h then concentrated by evaporation and spray-dried to obtain a fine brown powder (QF808, the active ingredient of VimangR, which melts at 215 – 210 jC with decomposition). We also assayed the effects of mangiferin, a C-glucosylxanthone (1,3,6,7-tetrahydroxyxanthone-C2-b) that is present in significant quantities in Vimang [16]. Mangiferin was obtained from the crude bark extract as described [16]. Vimang and mangiferin were dissolved in saline for in vivo administration and in dimethylsulfoxide (DMSO) for in vitro experiments. The concentrations used were between 0.1 and 100 Ag/ml for in vitro experiments and between 50 and 250 mg/kg b.w. for in vivo experiments. 2.3. Stimuli, drugs, and chemicals

2. Materials and methods 2.1. Animals Male Wistar rats (age 8– 10 weeks) (Iffa-Credo), purchased from Criffa (Barcelona, Spain), were used. They were housed (groups of five) in Macrolon cages (Panlab, Barcelona, Spain) on poplar shaving bedding (B&K Universal, G. Jordi, Barcelona, Spain) in a standard bioclean animal room, illuminated from 0800 to 2000 h (12 h light:12 h dark cycle) and maintained at a temperature of 22 –24 jC. The animals had free access to food pellets (B&K Universal) and to tap water, and were allowed to acclimatize for 1 week before the experiments. All experiments were carried out in accordance with European regulations on animal protection (Directive 86/609), the Declaration of Helsinki and/or the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health (NIH Publication no. 85-23, revised 1996). All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Santiago de Compostela.

Kluyveromyces lactis (strain NRRL-41140) cells were cultured on YPD medium (Difco, USA) at 30 jC, at a growth rate of 0.07 h 1, then washed with distilled water and finally freeze-dried. Stock solution (2 mg/ml) of phorbol 12-myristate 13-acetate (PMA) (Sigma, St. Louis, MO, USA) was dissolved in dimethylsulfoxide (DMSO) and stored in the dark at 80 jC until use. Stock solution of lipopolysaccharide (LPS) from Escherichia coli serotype 0111:B4 (Sigma) was made up at 10 Ag/ml in phenol-red-free Dulbecco’s Eagle medium (DMEM; Sigma) and stored at 20 jC until use. Thioglycollate broth (Merck, Germany) was prepared to a concentration of 3% (w/v) in PBS, autoclaved at 121 jC for 10 min, and stored at room temperature until use. Resveratrol, a naturally occurring polyphenol, was used for comparison in some experiments. Stock solutions of resveratrol (RESV, Sigma) and the arginine analogue N-monomethyl-L-arginine monoacetate (L-NMMA; Calbiochem, USA) were made up at 100 mM in DMSO (RESV) or water (L-NMMA). Both solutions were stored until use at 20 jC in the dark.

D. Garcı´a et al. / International Immunopharmacology 2 (2002) 797–806 L -Glutamine, sulfanilamide and naphthylenediamine hydrochloride were likewise purchased from Sigma, and recombinant murine gamma interferon (IFNg) from Genzyme, USA.

2.4. Isolation of rat peritoneal-exudate macrophages For induction of inflammatory responses, rats were injected intraperitoneally with 1 ml of 3% thioglycollate broth, and peritoneal exudate was extracted 5 days later. Rat resident and inflammatory peritoneal macrophages were obtained from rats euthanized by cervical dislocation in a laminar flow chamber to ensure sterile conditions. The abdomen of the rat was soaked with 70% ethanol for disinfection, a midline incision was then made with scissors, and the abdominal skin retracted. 30 ml de Hanks’ balanced salt solution (HBSS) was then injected into the peritoneal cavity using a syringe with a 19-G needle. After gentle abdominal massage, about 30 ml of peritoneal fluid was extracted using the same syringe and transferred to 50-ml sterile polypropylene tubes on ice. A 20 Al aliquot was then extracted for cell counting in a hemocytometer. The remaining cells were washed once by centrifugation at 400  g for 10 min at 4 jC and resuspended to a concentration of 106 cells ml 1. The number of viable cells was estimated by the trypan blue exclusion test: trypan blue (0.4% in PBS) was added to wells and incubated for 3 min at room temperature, after which the number of unstained (viable) and stained (nonviable) were counted. Aliquots of 100 Al of the cell suspension were added to the wells of 96well microculture plates (Corning, USA) or placed on microscope slides, and left for 90 min in a humidified incubator (37 jC, 5% CO2) to allow adhesion. Nonadherent cells were then removed by gently washing with HBSS. More than 97% of the adherent cells showed nonspecific esterase activity, determined as per Ref. [23], indicating that they were macrophages. 2.5. Phagocytic activity assay Phagocytic activity was characterized by the fluorimetric method described by Ref. [24], based on assay of the ingestion of yeast cells labeled with fluorescein isothiocyanate (FITC). To couple FITC, yeast cells were resuspended at 109 ml 1 in 50 mM sodium carbonate –bicarbonate

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buffer (pH 9.5) containing 150 mM NaCl and 40 mg of FITC. After incubation for 1 h at room temperature, the yeast suspension was centrifuged at 10,000  g for 5 min several times, each time discarding the supernatant and resuspending the pellet in PBS, until fluorescence in the supernatant dropped to zero. Fluorescence was measured in a microplate fluorescence reader (Bio-Tek Instruments, UK) with excitation at 490 nm and measurement at 525 nm. For phagocytic assay, an aliquot of 100 Al of phenol-red-free Dulbecco’s Eagle medium containing 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (Serva, USA), 5  107 FITC-labeled yeast cells and the required concentration of Vimang or mangiferin was added to each well of a 96-well microculture plate with adherent cells (106 per well) and incubated for 140 min at 37 jC under 5% CO2. The wells were then washed several times with PBS, and the cells were solubilized by adding 100 Al of 25 mM Tris – HCl pH 8.5 containing 0.2% sodium dodecyl sulfate. Fluorescence was measured in a microplate fluorescence reader (Bio-Tek Instruments) with excitation at 490 nm and measurement at 525 nm. The Vimang and mangiferin concentrations used were 100, 50, 10, 1.0, and 0.1 Ag/ml for in vivo assays with resident and thioglycollate-elicited macrophages, and 250, 100, and 50 mg/kg b.w. for the in vivo assays (by oral administration in a volume of 0.4 ml). 2.6. Chemoattraction assay To evaluate the effects of Vimang and mangiferin on the migration of macrophages to the peritoneum in response to intraperitoneal injection of a chemoattractant, we obtained hemocytometer counts of macrophages (i.e. cells showing nonspecific esterase activity) in the peritoneal exudate of rats stimulated intraperitoneally 5 days previously with 1 ml of 3% thioglycollate broth and 0.5 ml of Vimang or mangiferin at doses of 50, 100, or 250 mg/kg b.w. We also counted the total peritoneal-exudate cell population, allowing estimation of the proportion of macrophages in this population. 2.7. Assay of nitrite production A total of 100-Al aliquots of macrophages prestimulated in vivo with thioglycollate were incubated in

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microplates (Corning) at 37 jC with 5% CO2 for 90 min. After adhesion, the cells were gently washed with HBSS, then incubated for 48 h with 100 Al of phenolred-free DMEM containing 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (Serva), 10 U/ml of IFNg and 100 ng/ml of LPS. In some wells, the arginine analogue L-NMMA (250 AM) or resveratrol (25 Ag/ml) was included in the medium. Nitrite production in the culture supernatants was assayed by the Griess reaction, as described by Ref. [25], by measurement of the total amount of inorganic nitric oxides (NO). A total of 100-Al aliquots were removed from the medium and incubated with an equal volume of Griess reagent (1% sulfanilamide and 0.1% naphthylenediamine hydrochloride in 2.5% H3PO4), for 10 min at room temperature, and absorbance was measured at 530 nm in an ELISA reader (Titertek Multiscan, Flow Laboratories, Finland). Nitrite concentration was calculated with reference to a standard curve obtained using NaNO2 (1 –200 AM in culture medium). The concentrations tested were 100, 50, 10, and 1 Ag/ml for Vimang and 50, 10, and 1 Ag/ml for mangiferin. 2.8. Assay of ROS production The extracellular production of ROS during the respiratory burst response of macrophages was evaluated using OxyBURSTR Green Probes (Molecular Probes, the Netherlands). This reagent (OxyBURSTR Green H2HDD BSA reagent [bovine serum albumin (BSA) coupled to dihydro-2V,4,4,6,7,7V-hexafluorofluorescein (H2HFF)]) emits fluorescence when oxidized by the ROS produced during the respiratory burst [26]. A 1 mg/ml stock solution of the reagent was made up in HBSS containing 2 mM sodium azide and stored at 4 jC in the dark. For the assay, macrophages were incubated with the reagent at 10 Ag/ml in the wells of 96-well flat-bottom microtiter plates for 2 min at 37 jC, after which the corresponding concentration of Vimang or mangiferin was added. Finally, macrophages were stimulated by addition of 10 Ag/ml of PMA. Fluorescence was measured every 5 min for 45 min in a microplate fluorescence reader (Bio-Tek Instruments) with excitation at 490 nm and measurement at 525 nm. The rate of extracellular release of ROS was expressed as change in fluorescence per unit time (in arbitrary fluorescence units (AFU)) [27]. For

these assays, we used resident macrophages and thioglycollate-elicited macrophages (i.e. macrophages from rats that had received thioglycollate broth intraperitoneally 5 days previously). In some assays, L(+) ascorbic acid (100 AM) was added as inhibitory reference drug. Vimang was evaluated at concentrations of 100, 50, 10, and 1 Ag/ml, and mangiferin at 50, 10, and 1 Ag/ml. 2.9. Statistical analysis Results shown in the text and figures are expressed as means F standard error (S.E.M.). Means were compared by unpaired two-tailed Student’s t-tests. pValues less than 0.05 were considered significant.

3. Results 3.1. Effects of Vimang and mangiferin on the chemotaxis and phagocytic activity of macrophages We first investigated the effect of intraperitoneal administration of different doses of Vimang and mangiferin on the chemoattraction of macrophages by thioglycollate administered intraperitoneally 5 days previously. As shown in Fig. 1A, the administration of thioglycollate led to an increase in the number of macrophages in peritoneal exudate (indicating macrophage migration to the peritoneum). Intraperitoneal administration of Vimang or mangiferin partially inhibited this response (Fig. 1A) although neither caused a statistically significant reduction in the proportion of macrophages in the total peritoneal-exudate cell population (Fig. 1B). At the highest dose tested (250 mg/kg b.w.), macrophage count was reduced by 93% by Vimang and by 55% by mangiferin. We next evaluated the effects of Vimang and mangiferin on phagocytic activity. For these assays, we used FITC-labeled yeast cells (see Materials and Methods). First, we assayed phagocytosis by resident peritoneal macrophages, with Vimang or mangiferin present in the assay medium at 0.1 – 100 Ag/ml. Second, we assayed phagocytosis by peritoneal macrophages from rats that 5 days previously had received intraperitoneal injections of thioglycollate and Vimang or mangiferin at 50 –250 mg/kg b.w. In both cases, phagocytic activity was signifcantly reduced by both

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collate 5 days previously. They were then stimulated in vitro for 48 h with LPS and IFNg in the presence or absence of Vimang or mangiferin. L-NMMA at 250 AM (62.5 Ag/ml) was used as reference inhibitor of NO production. For comparative purposes, we also performed assays with resveratrol (100 AM, 25 Ag/ ml), a natural polyphenol with antioxidant properties [28]. As shown in Fig. 3, Vimang and mangiferin both significantly reduced nitrite production by activated macrophages, though this effect was not concentra-

Fig. 1. Macrophage counts in peritoneal exudate of rats 5 days after intraperitoneal inoculation with thioglycollate and Vimang or mangiferin at 50 – 250 mg/kg b.w. (A) Number of macrophages per ml of exudate. (B) Proportion of macrophages in the total peritonealexudate cell population. Values shown are means F S.E.M. of five determinations. Asterisks indicate significant differences ( * P < 0.01) with respect to the control (i.e. thioglycollate only).

Vimang and mangiferin (Fig. 2). The IC50% with resident macrophages was 87 Ag/ml for Vimang and 6.5 Ag/ml for mangiferin (Fig. 2B). The inhibition of phagocytosis was markedly higher with resident macrophages than with thioglycollate-elicited macrophages. 3.2. Effects of Vimang and mangiferin on RNS production by macrophages stimulated in vitro with LPS and IFNg To obtain the best possible NO response, macrophages were stimulated intraperitoneally with thiogly-

Fig. 2. In vitro and in vivo effects of Vimang and mangiferin on the phagocytosis of K. lactis cells (arbitrary fluorescence units; see Materials and Methods) by rat peritoneal macrophages. (A) Phagocytosis by resident peritoneal macrophages, with Vimang or mangiferin present in the assay medium at concentrations between 0.1 and 100 Ag/ml. (B) Phagocytosis by peritoneal macrophages from rats that 5 days previously had received intraperitoneal injections of thioglycollate (1 ml of 3% thioglycollate broth) and Vimang or mangiferin at doses between 50 and 250 mg/kg b.w. Values shown are means F S.E.M. Asterisks indicate significant differences ( ** P < 0.05; * P < 0.01) with respect to the corresponding control (i.e. no Vimang or mangiferin).

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was similar to that obtained with L(+) ascorbic acid (Fig. 4). To rule out the possibility that the observed partial inhibitions of phagocytosis, RNS production and ROS production were due to the presence of DMSO (used as a solvent), in all cases we ran parallel assays in which the medium contained DMSO alone at a 1/1000 dilution (the maximum concentration used in the assays). In no case was any inhibitory effect detected. Similarly, when DMSO (dilution 1/1000) was added to the culture medium, mean macrophage viability (determined on the basis of trypan blue exclusion: see Materials and Fig. 3. In vitro effects of Vimang and mangiferin on NO production in supernatants of 48-h cultures of rat peritoneal macrophages after stimulation in vivo with thioglycollate (1 ml of 3% thioglycollate broth) and in vitro with E. coli lipopolysaccharide (LPS; 100 ng/ml) and IFNg (10 U/ml), in the presence or absence of the iNOsinhibitor L-NMMA (250 AM) or the polyphenol resveratrol (100 AM). Values shown are means F S.E.M. of three determinations. Asterisks indicate significant differences ( * P < 0.01; ** P < 0.05) with respect to the control.

tion-dependent. The inhibitory effects of Vimang and mangiferin were weaker than those of L-NMMA, a nitric oxide synthase inhibitor, and similar to those of resveratrol (Fig. 3). 3.3. Effects of Vimang and mangiferin on ROS production by macrophages stimulated in vitro with PMA The effects of Vimang and mangiferin on ROS production by resident and thioglycollate-elicited macrophages in response to in vitro stimulation with PMA were investigated using the OxyBURST reagent. Vimang and mangiferin significantly reduced the PMA-induced ROS production by both resident and thioglycollate-elicited macrophages (Fig. 4A and B), and this inhibition was dose-dependent. Vimang reduced ROS production by thioglycollate-elicited macrophages (56 – 93%) less effectively than ROS production by resident macrophages (76 – 98%). Similar results were obtained with mangiferin, which induced an 88– 97% reduction in ROS production by resident macrophages and 68– 92% reduction in ROS production by thioglycollate-elicited macrophages. The reduction obtained with Vimang at 50 and 100 Ag/ml in elicited and resident macrophages

Fig. 4. In vitro effects of Vimang and mangiferin on extracellular ROS production (measured as the mean increase in fluorescence emission by oxidized OxyBURST Green H2HFF-BSA, in arbitrary units per minute) by rat resident peritoneal macrophages (A) and macrophages prestimulated in vivo with thioglycollate (B) in response to in vitro exposure to 10 Ag/ml of PMA, in the presence or absence of the ROS scavenger L(+) ascorbic acid (100 AM). Values shown are means F S.E.M. of three determinations. Asterisks indicate significant difference ( * P < 0.01; ** P < 0.05) with respect to the control.

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Methods) was 97 F 2% after 30 min, and 95 F 4% after 2 h (n = 5). To rule out the possibility that the observed partial inhibitions of phagocytosis, RNS production and ROS production were due to effects of the extracts on macrophage viability, Vimang or mangiferin was added to the culture medium (at 100 mg/ml, the maximum concentration used in the assays). In the case of Vimang, mean macrophage viability was 96 F 3% after 30 min, and 93 F 4% after 2 h (n = 5). In the case of mangiferin, mean macrophage viability was 97 F 2% after 30 min and 95 F 3% after 2 h (n = 5).

4. Discussion To evaluate the immunomodulatory effects of Vimang and mangiferin on rat macrophage functionality, we investigated effects on phagocytosis, chemotaxis, and respiratory burst activity. Although phagocytosis is an important process in host defense, through participation in antigen processing, cytokine production, and microbicidal and tumoricidal activities [29], phagocytic cells have also been implicated in various immunopathological processes, causing inflammation and destruction of tissues [30]. In such cases, it is important to reduce macrophage activation in order to attenuate the pathophysiological effects of inflammation. The chemoattraction and accumulation of macrophages is one of the initial steps in the inflammatory response. In this paper, we demonstrate that Vimang and mangiferin partially inhibit both the chemoattraction of macrophages in response to inflammatory stimuli and the phagocytosis of yeast cells. Chemotaxis is a process controlled by adhesion molecules and cytokines. Vimang and mangiferin are known to inhibit the expression and activity of the ICAM-1 receptor [31] and this possibly explains the inhibition of chemoattraction observed in the present study. RNS (including NO) and ROS (including O2 ) are important mediators in many inflammatory disorders [32]. The inflammatory effects of NO seem to be mediated by free radical mechanisms and to be associated with the expression of the inducible form of NO synthase (iNOS) [33]. Many cells have the ability to express iNOS, including macrophages, neutrophils,

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hepatocytes, and smooth muscle cells. In macrophages, iNOS can be expressed in response to various cytokines, including IFN-g, or bacterial endotoxins such as lipopolysaccharide (LPS). NO has several biological functions, including vasodilator and antithrombotic functions [34], and contribute to the immune defense against viruses and bacteria. However, excess production of NO is associated with various diseases such as diabetes, arthritis and other chronic inflammatory diseases, autoimmune diseases, septic shock, and atherosclerosis [35 – 39]. In this study, we have demonstrated that Vimang and mangiferin reduce NO production in peritoneal macrophages stimulated with LPS and IFNg. The inhibitory effects of Vimang and mangiferin are similar to these of resveratrol, a natural polyphenol that inhibits NO production and reduces the amount of cytosolic iNOS protein by a post-transcriptional mechanism [40,41]. This finding suggests that polyphenols present in the M. indica extracts, and especially mangiferin, may be responsible for the observed inhibition of NO production. The exposure of macrophages to appropriate stimuli activates a metabolic pathway known as the respiratory burst, characterized by the production of microbicidal oxidants (ROS) through the partial reduction of oxygen. O2 (one of the most important ROS) is produced through the interrelated pathways of calcium mobilization and protein kinase C activation by macrophages or neutrophils [42]. The key to this metabolic pathway is the respiratory burst oxidase, a membrane-bound enzyme that catalyzes the single electron reduction of oxygen to O2 at the expense of NADPH [4]. Excessive production of this species can lead to toxic reactions and potentiate several pathological conditions. The unsaturated fatty acid components of the cell wall are a major target, readily reacting with ROS to accept an extra electron which induces covalent interactions between neighboring molecules, causing severe disruption to membrane function. ROS may also damage nucleic acids, proteins, and carbohydrates. In this study, we stimulated resident peritoneal and thioglycollate-elicited macrophages with PMA, the fast activator of PKC [43] that is frequently used to trigger extracellular production of ROS (especially O2 and H2O2) by peritoneal macrophages. It has been reported that in phagocytic cells PMA activates protein kinase C, which is then translocated to the cell membrane,

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where it activates the NADPH-oxidase, which catalyzes the vectorial synthesis of O2 from oxygen and cellular NADPH supplied by the hexose monophosphate shunt [44]. Large amounts of these ROS are immediately released to the extracellular space and may react with the OxyBURST Green reagent to produce fluorescence. The present results confirm the potent antioxidant properties of Vimang and mangiferin, which both significantly reduced extracellular ROS production by peritoneal and thioglycollate-elicited macrophages stimulated in vitro with PMA. The inhibitory effect of Vimang at concentrations of 50 and 100 Ag/ml was similar to that of ascorbic acid, a known antioxidant compound. These inhibitory effects are probably due to ROS-scavenging properties of the extract. Previously reported antioxidant effects of Vimang include efficient in vitro scavenging of OH and HOCl, and inhibition of the peroxidation of rat-brain phospholipids, of the DNA damage caused by bleomicin and the copperphenantroline system, and of H2O2 production by peritoneal macrophages stimulated in vivo with TPA [10,11]. It has previously been reported that Vimang reduces the production of TNFa in macrophage cell line RAW264 stimulated with proinflammatory stimuli (LPS 100 Ag/ml; IFNg 10 U/ml) [31]. TNFa is a proinflammatory cytokine and its levels in plasma are directly correlated with the ability of phagocytes to generate superoxide and to increment the activity of iNOS and, thus, NO levels [45,46]. In patients with rheumatoid arthritis, treatment with anti-TNFa antibodies reduced NOS activity and disease severity [47]. Accordingly, the inhibitory effects of Vimang on ROS and NO production might be related to inhibition of TNFa production by macrophages. Various hypotheses can be put forward to explain the mechanism/s of the observed modulation of macrophage function by Vimang (which the present results suggest to be largely due to its major polyphenol, mangiferin). First, mangiferin may, in some way, block the action of TNFa, which induces the expression of macrophage chemotactic protein 1 (MCP1). Second, it may, in some way, affect the production of immunomodulatory cytokines (e.g. IFNg, IL-4, IL-10) that are thought to have potent effects on macrophage activation [48]. Third, polyphenols like mangiferin are reducing agents that function as antioxidants, by virtue

of the hydrogen-donating activity of their phenolic hydroxyl groups [49] and, thus, may directly inhibit the macrophage respiratory burst, due to ROS-scavenging effects [50]. Fourth, polyphenols have been reported to act as chelators of metals [51] including Ca2 + , which is involved in cellular activation processes including the mannose-specific interaction between macrophages and yeast cells during phagocytosis [52] and the (protein kinase C)-mediated phosphorylation occurring during superoxide production [53,54]. In conclusion, Vimang is an extract capable of modulating the activation and functionality of macrophages, through partial inhibition of chemotactic migration, phagocytic activity, ROS production and RNS production. Mangiferin, a glucosylxanthone present in the extract, contributes to this effect. We suggest that Vimang might be used in disorders characterized by overactivation of macrophages such as rheumatoid arthritis and other inflammatory diseases. Moreover, Vimang contains between 0.03 and 0.08% selenium [16]. Selenium is an essential component of the enzyme glutathion peroxidase, an important antioxidant enzyme that catalyzes the reduction of hydroperoxides produced from oxidized species such as superoxide and lipoperoxides [55]. As selenium concentrations are relativity low in the serum of patients with rheumatoid arthritis [56], Vimang might be of value as a supplementary source of selenium in these patients.

Acknowledgements This work was financially supported by grant PGID-T99MAR20301 from the Xunta de Galicia, Spain. Dagmar Garcı´a is supported by a fellowship from the Agencia Espan˜ola de Cooperacio´n Internacional (AECI), Spain. The authors are grateful to Dr. Marı´a Isabel Gonza´lez Siso of the Biochemistry Laboratory, University of A Corun˜a (Spain), for the generous supply of Kluyveromyces lactis cells.

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