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

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 microg/ml reduced the phagocytosis of yeasts cells by resident peritoneal and thioglycollate-elicited macrophages; (c) in vitro concentrations of 1-50 microg/ml reduced nitric oxide (NO) production by thioglycollate-elicited macrophages stimulated in vitro with lipopolysaccharide (LPS) and IFNgamma; and (d) in vitro concentrations of 1-50 microg/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 depressor 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.     

Tumor cytotoxicity of peritoneal macrophages induced by OK-432

In the present study we investigated the enhancement of cytotoxicity of peritoneal macrophages induced by OK-432. Rats received an i.p. injection of OK-432 at doses of 0.1, 0.5 or 1.0 KE/rat. Two days later, rats were sacrificed and peritoneal macrophages were isolated. Then the number of macrophages was counted, and the macrophages were analyzed for their lactic dehydrogenase (LDH) activity, acid phosphatase (ACP) activity, phagocytic activity, secretion of nitric oxide (NO) and cytotoxicity. The number of peritoneal macrophages, the activity of LDH and ACP, phagocytic activity, NO secretion, and cytotoxicity were increased with the increasing doses of OK-432. The results suggested that OK-432 enhanced tumor cytotoxicity of peritoneal macrophages by three steps. The first step is to attract a great number of macrophages into the peritoneal cavity. The second step is to enhance the phagocytic and eliminating function of these macrophages. The last step is to increase the non-contact cytotoxicity of macrophages.     

Effect of the essential oil from the flowers of Magnolia sieboldii on the lipopolysaccharide-induced production of nitric oxide and prostaglandin E2 by rat peritoneal macrophages

The essential oil from the flowers of Magnolia sieboldii was tested for its effects on lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) by rat peritoneal macrophages. It was shown to induce the production of NO and PGE 2 in a concentration-dependent manner (3 - 30 microg/ml). Gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) led to the identification of sixty compounds, of which beta-elemene (18.0 %), alpha-terpinene (14.83 %) and beta-myrcene (12.72 %) were the major constituents. Among these three compounds, alpha-terpinene was found to be the most effective one with inhibitory activity on NO and PGE(2) production by LPS-stimulated rat peritoneal macrophages.     

Protection against septic shock and suppression of tumor necrosis factor alpha and nitric oxide production on macrophages and microglia by a standard aqueous extract of Mangifera indica L. (VIMANG). Role of mangiferin isolated from the extract.

The present study illustrates the effects of a standard aqueous extract, used in Cuba under the brand name of VIMANG, from the stem bark of Mangifera indica L. on the production of tumor necrosis factor alpha (TNFalpha) and nitric oxide (NO) in in vivo and in vitro experiments. In vivo was determined by the action of the extract and its purified glucosylxanthone (mangiferin) on TNFalpha in a murine model of endotoxic shock using Balb/c mice pre-treated with lipopolysaccharide (LPS) 0.125 mg kg(-1), i.p. In vitro, M. indica extract and mangiferin were tested on TNFalpha and NO production in activated macrophages (RAW264.7 cell line) and microglia (N9 cell line) stimulated with LPS (10ng ml(-1)) and interferon gamma (IFNgamma, 2U ml(-1)). M. indica extract reduced dose-dependently TNFalpha production in the serum (ED50 = 64.5 mg kg(-1)) and the TNFalpha mRNA expression in the lungs and livers of mice. Mangiferin also inhibited systemic TNFalpha at 20 mg kg(-1). In RAW264.7, the extract inhibited TNFalpha (IC50 = 94.1 microg ml(-1)) and NO (IC50 = 64.4 microg ml(-1)). In microglia the inhibitions of the extract were IC50 = 76.0 microg ml(-1) (TNFalpha) and 84.0 microg ml(-1) (NO). These findings suggest that the anti-inflammatory response observed during treatment with M. indica extract must be related with inhibition of TNFalpha and NO production. Mangiferin, a main component in the extract, is involved in these effects. The TNFalpha and NO inhibitions by M. indica extract and mangiferin on endotoxic shock and microglia are reported here for the first time.     

Effects of mancozeb on the activities of murine peritoneal macrophagesin vitro andex vivo

Mancozeb (MCZ) is known to have detrimental effects on the reproductive system, but the toxicity of MCZ on immune responses has not been systematically investigated. We investigated the effects of MCZ exposure on the activities of murine peritoneal macrophages through evaluation of MCZ-induced alteration of nitric oxide (NO) production and tumor necrosis factor-alpha (TNF-alpha) synthesis. Macrophages were examined ex vivo from mice orally treated with various doses of MCZ for 5 consecutive days per week for 4 weeks (subacute exposure, 250, 1000, 1500 mg/kg/day) followed by culture for 2 (TNF-alpha) or 3 days (NO) in the presence of LPS plus IFN-gamma. Macrophages from naive mice were also cultured with various concentrations of MCZ (0.05, 0.25, 0.5, 1 and 2 microg/mIL in the presence of LPS plus IFN-gamma for 2 (TNF-alpha) or 3 days (NO) in vitro. NO production was decreased with the in vitro exposure to all concentrations of MCZ. However, the amount of NO production by peritoneal macrophages from MCZ-subacutely exposed mice was increased in comparision with that of control group. In vitro, MCZ suppressed TNF-alpha secretion with significant reduction at 2 microg/mL MCZ. Conversely, TNF-alpha release was enhanced ex vivo. This study provides the substantial evidence on MCZ-induced alternation in macrophage activity. In order to clearly understand the contrasting effect of MCZ on peritoneal macrophage activity, it is necessary to further investigate the influence of major metabolite of MCZ (ETU) exposure on the NO production and TNF-alpha synthesis.     

Inhibition of inducible nitric oxide synthase by beta-lapachone in rat alveolar macrophages and aorta

Beta-lapachone, a plant product, has been shown to be a novel inhibitor of DNA topoisomerase. In this study, we performed experiments to examine the effects of beta-lapachone on lipopolysaccharide (LPS)-induced inducible nitric oxide (NO) synthase (iNOS) in rat alveolar macrophages and aortic rings. In alveolar macrophages, incubation with LPS (10 microg ml(-1)) for various time intervals resulted in a significant increase in nitrite production and iNOS protein synthesis, that was inhibited by coincubation with beta-lapachone (1-4.5 microM) without any cytotoxic effects. However, addition of beta-lapachone after induction of NO synthase by LPS failed to affect the nitrite production. Treatment with LPS (10 microg ml(-1)) for 6 h resulted in significant expression of mRNA for iNOS which was significantly inhibited in the presence of beta-lapachone (3 microM) in alveolar macrophages. In endothelium-intact rings of thoracic aorta, beta-lapachone (1 and 3 microM) markedly inhibited the hypocontractility to phenylephrine in aortic rings treated with LPS (10 microg ml(-1)) for 4 h. When beta-lapachone was added 3 h after LPS into the medium, the contractions evoked by phenylephrine were not significantly different in the presence or absence of beta-lapachone. Treatment with LPS (10 microg ml(-1)) for 4 h resulted in a significant increase in iNOS protein synthesis which was inhibited in the presence of beta-lapachone (3 microM), but did not affect the constitutive (endothelial and neuronal) NOS forms in aortic rings. These results indicate that beta-lapachone is capable of inhibiting expression and function of iNOS in rat alveolar macrophages and aortic rings. It is considered that beta-lapachone can be developed as a potential anti-inflammatory agent in the future.     

Anti-inflammatory effects of the methanol extract of Sedum telephium ssp. maximum in lipopolysaccharide- stimulated rat peritoneal macrophages

Sedum telephium ssp. maximum is a medicinal plant that possesses anti-inflammatory, analgesic and keratolytic properties. We investigated the anti-inflammatory activity of its methanolic extract (STME) in rat peritoneal macrophages (MPhis) stimulated with lipopolysaccharide from Salmonella enteritidis. After stimulation with 10 microg/ml of LPS, MPhis were coincubated with different doses of STME (8, 16 and 32 microg/ml) or RPMI medium alone using different times of incubation. STME reduced levels of tumor necrosis factor-alpha, both mRNA and its protein, and significantly decreased IL-1beta and IL-6 production. Moreover, STME inhibited inducible nitric oxide synthase expression and blunted nitrite release and inhibited both extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase activation in lipopolysaccharide-stimulated MPhis. Data show that STME might be useful as a potential anti-inflammatory agent.     

Effects of NO2 on immune responses

The effects of NO2 on immune responses of mice were investigated. Mice were exposed to various concentrations of NO2 in inhalation chambers. After exposure the following parameters were measured: phagocytosis of polystyrene beads by both peritoneal and alveolar macrophages, production of antibody-forming cells from mice immunized with sheep erythrocytes, lymphocyte blastogenesis of splenic cells, and susceptibility to influenza virus. The production of antibody-forming cells was reduced in mice that were exposed to 5 ppm NO2. The serum antibody titers, phagocytosis, and other immune parameters measured were not affected. Exposure to NO2 did not affect mortality to influenza virus. These data indicate that certain immune parameters were altered by exposure to NO2; however, NO2 does not appear to be a major immunosuppressive factor at the concentrations tested.     

Neuraminidase inhibitors reduce nitric oxide production in influenza virus-infected and gamma interferon-activated RAW 264.7 macrophages

The influenza virus (influenza) infection causes an intense infiltration of pulmonary tissues by macrophages, which abundantly generate a free radical, nitric oxide (NO) resulting in lung damage. Neuraminidase inhibitors (NIs) restrict influenza virus replication but whether they can suppress NO production within macrophages is unknown. RAW 264.7 macrophages were exposed to interferon-gamma (IFN-gamma), live influenza (A/PR/8/34) or a combination of both and were treated with NIs (oseltamivir or zanamivir). Results revealed that the drugs reduced a synergy between influenza and IFN-gamma in NO synthesis within the cells at all of the used concentrations (0.01, 0.1, 1 microg/ml). In contrast to zanamivir, this effect occurred in a concentration-dependent manner with oseltamivir treatment. On the other hand, all concentrations of zanamivir significantly suppressed NO production in comparison to that upon the combined exposure only (p < 0.05). Both compounds also considerably decreased NO generation in the IFN-gamma-stimulated macrophages, and zanamivir in the influenza-infected cells as well. However, neither of the drugs inhibited iNOS mRNA expression in the cells containing these stimulants. Additionally, the data indicate that a prodrug oseltamivir can be activated in vitro within the macrophage cultures. These findings are important for designing treatment approaches to limit pulmonary inflammation during influenza infection.     

酵母多糖对小鼠腹腔巨噬细胞产生一氧化氮和白细胞介素-1的影响

目的探讨酵母多糖对小鼠腹腔巨噬细胞产生一氧化氮 (NO)和白细胞介素 1(IL 1)的影响。方法将不同剂量的酵母多糖加入体外培养的小鼠腹腔巨噬细胞中 ,取细胞培养上清液根据Griess反应检测NO-2 的量 ,间接反映巨噬细胞产生NO的生成量 ,并用溴化四唑蓝 (MTT)比色法检测上清液中IL 1的生成量。结果酵母多糖可明显促进小鼠腹腔巨细胞产生NO和IL 1,NO的生成量呈现剂量依赖关系。结论酵母多糖可诱导小鼠腹腔巨噬细胞产生NO和IL 1,可能是酵母多糖调节机体免疫功能、杀伤病原微生物和抗肿瘤的重要途径     

Suppression of NO production in activated macrophages in vitro and ex vivo by neoandrographolide isolated from Andrographis paniculata

In this study, we investigated the in vitro and ex vivo suppressive effects of Andrographis paniculata on nitric oxide (NO) production in mouse peritoneal macrophages elicited by bacillus Calmette-Guéin (BCG) and stimulated by lipopolysaccharide (LPS). Incubation of BCG-induced macrophages with the methanol extract of A. paniculata reduced LPS stimulated NO production. The diterpene lactones andrographolide and neoandrographolide were isolated as active components from the extract. These compounds suppressed NO production in a concentration-dependent manner in the concentration range from 0.1 to 100 microM and their IC50 values were 7.9 and 35.5 microM. Neoandrographolide also suppressed NO production by 35 and 40% when the macrophages were collected after oral administration of neoandrographolide at doses of 5 and 25 mg/kg/d and LPS stimulated NO production was examined. However, andrographolide did not reduce NO production on oral administration at the same doses. These results indicate that neoandrographolide, which inhibited NO production both in vitro and ex vivo may play an important role in the use of A. paniculata as an anti-inflammatory crude drug.     

In vitro induction of nitric oxide by mouse peritoneal macrophages treated with human placental extract

Nitric oxide (NO) is an important cellular mediator of tissue repair. It is produced in macrophages by the enzyme inducible nitric oxide synthase (iNOS) during wound healing. An aqueous extract of human placenta used as wound healer, has been investigated in terms of induction of NO by mouse peritoneal macrophages as well as human monocyte derived macrophages. NO production was estimated in macrophages culture supernatants. Incubation of 0.1 to 20 mg/ml of placental extract with 2x10(6) cells in vitro produced 10 to 100 microM of nitrite (n=4) in a dose dependent manner suggesting production of NO. With increase of NO production, NADPH present in the applied extract decreased proportionately. Application of L-NG monomethyl arginine (L-NMMA), an NO synthase (NOS) inhibitor, reduced the production of NO at the basal level. Dose dependent release of IFN-gamma with respect to placental extract by the mouse macrophages was observed. It has been observed that human monocytes derived macrophages also produced significant amount of NO by induction of the extract. Similar induction of NO by placental extract in presence and absence of polymyxin B suggested that this property is not likely to be mediated by the endotoxin/LPS.     

羊胎盘免疫调节因子对小鼠腹腔巨噬细胞免疫功能的影响

目的探讨羊胎盘免疫调节因子(GPIF)对小鼠腹腔巨噬细胞免疫功能的影响。方法采用巨噬细胞体外培养的方法。分3个实验组,对腹腔巨噬细胞分别给予0 .0 5 ,0 .1和0 .5mg/mlGPIF进行体外培养,培养结束后,测定腹腔巨噬细胞吞噬中性红的能力、对溴化四唑蓝(MTT)的还原能力,以及分泌一氧化氮(NO)和白细胞介素 1(IL-1)的量。结果与对照组比较,各浓度GPIF均有促进腹腔巨噬细胞吞噬中性红能力作用,且能显著提高腹腔巨噬细胞对MTT的还原能力,增加NO和IL-1的分泌量(P <0 .0 5 )。结论GPIF具有非特异性免疫增强作用     

香菇多糖对巨噬细胞一氧化氮和一氧化氮合酶活性的影响

目的研究香菇多糖(LTN)诱导巨噬细胞的一氧化氮(NO)生成和一氧化氮合酶(iNOS)的活性,探讨LTN的免疫调节作用机理.方法采用Griess反应和荧光法测定不同剂量的LTN作用小鼠腹腔巨噬细胞后NO的生成量和iNOS活性.观察mRNA转录抑制剂、蛋白质合成抑制剂和iNOS抑制剂对巨噬细胞NO的生成和iNOS活性的影响.结果LTN能使小鼠腹腔巨噬细胞NO生成增加,iNOS活性增高,并呈作用剂量依赖关系.3种抑制剂均能抑制LTN诱导的小鼠腹腔巨噬细胞N0的生成和iNOS活性.结论LTN能刺激小鼠腹腔巨噬细胞提高iNOS活性和NO的生成.提示LTN的免疫调节作用机制可能与LTN刺激巨噬细胞NO生成有关.     

Augmentation of macrophage phagocytosis by modified arabinoxylan rice bran (MGN-3/biobran)

MGN-3/Biobran, modified arabinoxylan rice bran, has been shown to be a potent biological response modifier (BRM) as manifested by stimulation of different arms of the immune system such as NK, T and B cells; however, its effect on macrophages has not yet been studied. The effects of MGN-3 on macrophage function was examined in vitro using 3 models: human macrophage cell line U937, murine macrophage cell line RAW264.7, and murine peritoneal macrophages (P-M phi). Treatment with MGN-3 resulted in an increase in the percentages of attachment and phagocytosis of yeast by macrophages. The effect depends on the type of macrophage and the dose of MGN-3 applied. Macrophages also demonstrated enhancement in their spreading ability, post treatment with MGN-3. Results also showed that MGN-3, in a dose dependent manner (1, 10,100 microg/ml), significantly induced high levels of production of cytokines: TNF-alpha; and IL-6. In addition, MGN-3 significantly increased nitric oxide (NO) production. This data demonstrates that MGN-3 is a potent inducer of phagocytic function by macrophage, and suggests that MGN-3 is a useful agent for fighting microbial infection.     

Inhibitory effects of metabolite I of erdosteine on the generation of nitric oxide and peroxynitrite chemiluminescence by human neutrophils

Polymorphonuclear neutrophils (PMNs) can generate superoxide anions and nitric oxide (NO), which is not only an important mediator of various cellular activities, but can also react with superoxide anions to produce peroxynitrite anions (ONOO-). Peroxynitrite is a potent and potentially toxic oxidant that damages various types of biomolecules. It preferentially mediates the oxidation of thiolic groups in protein and non-protein molecules, thus altering their functions. The aim of this study was to examine whether, in addition to its ability to reduce the respiratory bursts of human PMNs, the SH metabolite I (Met I) of erdosteine, can interfere with NO and NO-derived peroxynitrite production, thus extending its antioxidant activity. This was done by means of the luminol amplified chemiluminescence (LACL), which has been widely used to detect the production of reactive oxidant species (ROS) by PMNs under various conditions. At 5 and 10 microg/ml, Met I significantly reduced LACL after fMLP and PMA stimulation. When L-Arg was added to the reaction medium, as a NO donor, the chemiluminescence of fMLP increased by up to 67% and that of PMA by up to 132%, but was once again significantly reduced by 5 and 10 microg/ml of Met I. In a cell-free system, the use of linsidomine (SIN-1) makes it possible to investigate the behavior of LACL induced by peroxynitrite release, which was significantly reduced by Met I concentrations ranging from 1.25 to 10 microg/ml. Our findings indicate that Met I, a molecule with a SH group, reacts with ROS, NO and NO-derived peroxynitrite, and has both antioxidant and scavenging activity. This is of interest for the strategy of protecting against damage induced by radical species in the pulmonary cell environment, in which they can induce a phlogogenic loop, and suggests that adding exogenous thiols may be useful in antagonizing the toxic effects of reactive molecules on endogenous thiols.     

Fucogalactan isolated from Sarcodon aspratus elicits release of tumor necrosis factor-alpha and nitric oxide from murine macrophages

Eight species of mushrooms were evaluated for mitogenic activity by the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method using spleen cells of C3H/HeN female mice. The hot water-soluble (HWS) fraction extracted from Sarcodon aspratus showed the highest activity. The mitogen in Sa. aspratus was isolated by Sepharose 6B and DEAE-Sepharose CL-6B column chromatography. Preliminary structural analyses indicated that the mitogen was a fucogalactan. Fucogalactan elicited the release of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in macrophages of mice in vitro. TNF-alpha production induced with 50 microg/ml of fucogalactan was significantly higher than that induced by lentinan (500 microg/ml) by approximately 4.3-fold. Also, fucogalactan showed dose dependence at concentrations from 5 to 500 microg/ml in NO production. Thus, fucogalactan does elicit the release of cytokines such as TNF-alpha and NO.