Mustard leaf suppresses nitric oxide synthesis by mouse macrophages

The effect of mustard leaf on nitric oxide (NO) production was investigated using lipopolysaccharide (LPS)-stimulated peritoneal macrophages. LPS induced the production of a large amount of nitrite, an indicator of NO. However, the addition of the ethylacetate (EtOAc) or n-butanol (BuOH) fraction of mustard leaf to LPS-stimulated peritoneal macrophages inhibited excessive production of nitrite significantly. Moreover, compared with no treatment, LPS impaired cell viability significantly. However, the EtOAc fraction of mustard leaf significantly protected cells that had been exposed to LPS. In addition, the result of nitrite production per cell indicated that the mustard leaf fractions significantly suppressed nitrite synthesis by macrophages. In particular, the EtOAc fraction was a stronger inhibitor of nitrite synthesis than the BuOH fraction. As NO is one of the critical mediators in various disorders, these results may help to explain certain pharmacological activities of mustard leaf.     

Bovine dialyzable leukocyte extract modulates the nitric oxide and pro-inflammatory cytokine production in lipopolysaccharide-stimulated murine peritoneal macrophages in vitro

Lipopolysaccharides (LPS) released from Gram-negative bacteria after infection initiate an exagerated response that leads to a cascade of pathophysiological events termed sepsis. Monocytes or macrophages produce many of the mediators found in septic patients. Targeting of these mediators, especially tumor necrosis factor (TNF)-alpha and nitric oxide (NO), has been pursued as a mean of reducing mortality in sepsis. Bovine dialyzable leukocyte extract (bDLE) is a dialysate of a heterogeneous mixture of low-molecular-weight substances released from disintegrated leukocytes of the blood or tissue lymphoid. In this study, to determine whether bDLE modulates NO and pro-inflammatory cytokine production, murine peritoneal macrophages were treated with bDLE (0.05 or 0.5 U/mL) before LPS (20 mg/mL) stimulation, and also LPS-stimulated murine peritoneal macrophages were treated with bDLE (0.05 or 0.5 U/mL) at 0, 4, 8, 12, and 24 hours. The bDLE significantly decreased NO production, and also decreased TNF-alpha and interleukin (IL)-6 but increased IL-10 production in LPS-stimulated murine peritoneal macrophages. Our results demonstrate that bDLE plays an important role in modulating TNF-alpha, IL-6, and NO production through IL-10, and this may offer therapeutic potential in clinical endotoxic shock.     

知母皂苷对脂多糖诱导巨噬细胞炎症介质释放影响

目的观察知母皂苷是否能对抗脂多糖(LPS)引起的小鼠腹腔巨噬细胞炎症介质释放并探讨丝裂原活化蛋白激酶(MARK)信号转导通路影响。方法实验设对照组,脂多糖组,知母皂苷低、中、高剂量组和阻断剂组,Griess法测定巨噬细胞培养上清液一氧化氮(NO)含量变化;酶联免疫吸附法测定巨噬细胞培养上清液中白介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)含量;免疫化学染色法观察诱导型一氧化氮合酶(iNOS)表达。结果加入脂多糖作用2 h,巨噬细胞上清液中NO含量明显增加,24 h达高峰,知母皂苷(10、30、100 μmol/L) 可抑制脂多糖引起的IL-1β、TNF-α、NO增加和iNOS表达增多,100 μmol/L 知母皂苷组IL-1β、TNF-α和NO水平分别从(698.8±32.0)ng/L、(257.4±27.3)ng/L和(181.0±19.9) μmol/L下降到(382.7±48.9)ng/L、(111.6±23.9)ng/L和(82.6±18.1)μmol/L;SP600125和SB203580均可不同程度抑制脂多糖引起的巨噬细胞IL-1β、TNF-α和NO增加;S-甲基异硫脲可完全对抗脂多糖引起NO释放。结论知母皂苷明显抑制脂多糖引起的巨噬细胞炎症因子释放,其机制与下调p38MAPK和c-Jun氨基末端激酶(JNK)信号通路表达有关。     

Endotoxin-Binding Peptides Derived from Casein Glycomacropeptide Inhibit Lipopolysaccharide-Stimulated Inflammatory Responses via Blockade of NF-κB activation in macrophages

Systemic low-grade inflammation and increased circulating lipopolysaccharide (LPS) contribute to metabolic dysfunction. The inhibitory effects and underlying molecular mechanisms of casein glycomacropeptide (GMP) hydrolysate on the inflammatory response of LPS-stimulated macrophages were investigated. Results showed that the inhibitory effect of GMP hydrolysates obtained with papain on nitric oxide (NO) production were obviously higher than that of GMP hydrolysates obtained with pepsin, alcalase and trypsin (p < 0.05), and the hydrolysate obtained with papain for 1 h hydrolysis (GHP) exhibited the highest inhibitory effect. Compared with native GMP, GHP markedly inhibited LPS-induced NO production in a dose-dependent manner with decreased mRNA level of inducible nitric oxide synthase (iNOS). GHP blocked toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway activation, accompanied by downregulation of LPS-triggered significant upregulation of tumor necrosis factor (TNF)-α and interleukin (IL)-1β gene expression. Furthermore, GHP could neutralize LPS not only by direct binding to LPS, but also by inhibiting the engagement of LPS with the TLR4/MD2 complex, making it a potential LPS inhibitor. In conclusion, these findings suggest that GHP negatively regulates TLR4-mediated inflammatory response in LPS-stimulated RAW264.7 cells, and therefore may hold potential to ameliorate inflammation-related issues.     

Naringenin more effectively inhibits inducible nitric oxide synthase and cyclooxygenase-2 expression in macrophages than in microglia

Macrophages and microglia are thought to account for initial disease progression in acute myocardial infarction and acute ischemic stroke. Before our study, the inhibitory effects of naringenin, a flavonoid, on lipopolysaccharide (LPS)-induced inflammation in macrophages and microglia have not been fully reported and compared. We hypothesized that naringenin can effectively inhibit LPS-induced inflammation of macrophages and microglia at different concentrations, the range of which is broader, with the lowest concentration more easily achieved in macrophages. In this study, we compared the anti-inflammatory effects of naringenin on LPS-stimulated RAW 274.6 macrophages and BV2 microglia and the suppression effects of naringenin and vitamin C (a well-known anti-inflammatory agent) on LPS-induced nitrite production. The results show that macrophages could maintain cell viability at higher naringenin concentrations and were more easily activated by LPS in comparison to microglia (200 vs 100 μmol/L; 0.1 vs 1 μg/mL). Under LPS (1 μg/mL) stimulation in both cell types, naringenin (up to 200 μmol/L in macrophages and 100 μmol/L in microglia) inhibited nitrite production and inducible nitric oxide synthase and cyclooxygenase-2 expression in a dose-dependent manner. The range of naringenin concentrations for inhibition was broader, and the lowest concentration was more easily achieved in macrophages; the lowest effective concentrations of naringenin to achieve constant suppression effect were 50 μmol/L in macrophages and 100 μmol/L in microglia, respectively. Vitamin C (100 μmol/L), compared with naringenin (100 μmol/L), had less and no suppression effect on LPS (1 μg/mL)-induced nitrite production in macrophages and microglia, respectively. In conclusion, naringenin more effectively inhibits the LPS-induced inflammatory status, including nitrite production and inducible nitric oxide synthase and cyclooxygenase-2 expression, in macrophages than in microglia. The findings of the present study suggest that consumption of naringenin-containing flavonoids might be beneficial to the cardiovascular and cerebrovascular inflammatory process.     

Antioxidative and anti-inflammatory effects of Saururus chinensis methanol extract in RAW 264.7 macrophages

Natural products are known to be sources of bioactive components exerting antioxidative and anti-inflammatory activities. We evaluated the suppressive effects of the methanol extract (0-45 microg/mL) of the aerial parts of Saururus chinensis (Lour.) Baill (Saururaceae) on lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production and oxidative stress buildup in the RAW 264.7 murine macrophages. Treatment of RAW 264.7 cells with S. chinensis methanol extract (SME) significantly reduced LPS-stimulated NO production in a concentration-dependent manner. Treatment with SME reduced thiobarbituric acid-reactive substances accumulation and enhanced glutathione levels and activities of antioxidative enzymes, including superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase, in LPS-stimulated macrophages compared with LPS-only treated cells. Expression of inducible NO synthase (iNOS) mRNA was also suppressed in SMEtreated cells. The specific DNA binding activities of nuclear factor kappaB (NFkappaB) on nuclear extracts from SME-treated cells were significantly suppressed. These results suggest that SME has antioxidative and anti-inflammatory activities by enhancing antioxidative defense systems and suppressing NO production via the down-regulation of iNOS expression and NFkappaB activity.     

Alcohol and Nitric Oxide Production by Cells of the Brain

The l-arginine–nitric oxide pathway is important to both physiological and pathologic brain events. Brain tissue contains cells able to express all known isoforms of nitric oxide synthase, the rate-limiting enzyme in nitric oxide (NO) production and release. Effects of ethanol on NO production may be important to ethanol modification of brain function. Recent studies support this idea and demonstrate diverse interactions. For example, acute ethanol treatment decreases NMDA- and cytokine-stimulated NO synthesis by cortical neurons and glia, respectively, but enhances cytokine-stimulated NO synthesis in blood–brain barrier endothelial cells and does not affect norepinephrine-stimulated NO synthesis in medial basal hypothalamus. Furthermore, chronic ethanol enhances NMDA-stimulated NO synthesis in cortical neurons, but more potently decreases cytokine-induced NO synthesis in glial cells. The mechanisms underlying these effects are partially understood and include changes in NOS-2 gene expression. These observations illustrate that ethanol selectively affects NO production by brain cells, which may relate to reported behavioral interactions, but the extend and direction of change depends on cell type and length of exposure.     

2,5-dihydroxyacetophenone isolated from Rehmanniae Radix Preparata inhibits inflammatory responses in lipopolysaccharide-stimulated RAW264.7 macrophages

Rehmanniae Radix Preparata, the steamed root of Rehmannia glutinosa Libosch, has been widely used for the treatment of inflammatory conditions in Oriental medicines. In this study we evaluated the effects of 2,5-dihydroxyacetophenone (DHAP) isolated from Rehmanniae Radix Preparata on inflammatory responses in lipopolysaccharide (LPS)-stimulated RAW264.7 mouse macrophages. LPS-stimulated RAW264.7 cells were used to investigate the anti-inflammatory activity of DHAP on the production of inflammatory mediators such as nitric oxide (NO), inducible NO synthase (iNOS), tumor necrosis factor-α (TNF-α), and interleukin (IL)-6. DHAP significantly inhibited NO production via the suppression of iNOS expression and significantly decreased levels of the pro-inflammatory cytokines TNF-α and IL-6 via the down-regulation of their mRNA expression in LPS-stimulated RAW264.7 cells. DHAP potently inhibited the phosphorylation of extracellular signal-related kinase (ERK) 1/2 and the nuclear translocation of nuclear factor-κB (NF-κB) p65 in LPS-stimulated cells. These results indicate that DHAP inhibits the production of inflammatory mediators in activated macrophages by blocking the ERK1/2 and NF-κB signaling pathways. Our results suggest that DHAP from Rehmanniae Radix Preparata has anti-inflammatory activity in activated macrophages, raising the possibility that this compound has a therapeutic potential for inflammatory conditions.     

Extracts of Actinidia arguta stems inhibited LPS-induced inflammatory responses through nuclear factor–κB pathway in Raw 264.7 cells

The inflammatory response protects our body from bacteria and tumors, but chronic inflammation driven by the persistent activation of macrophages can lead to serious adverse effects including gastrointestinal problems, cardiac disorders, and a sore throat. Part of the ongoing research is focused on searching for antiinflammatory compounds from natural sources, so we investigated the effects of hardy kiwis (Actinidia arguta, Lauraceae) stems on inflammation induced by lipopolysaccharide (LPS) in Raw 264.7 cells to test the hypothesis that antiinflammatory effects of A. arguta stems were exerted through the inhibition of the nuclear factor (NF)-κB pathway. The methanol extract of A. arguta (20 μg/mL) stems lowered nitric oxide production in LPS-stimulated Raw 264.7 cells by 40%. It was then partitioned with hexane, chloroform, ethyl acetate, butanol, and water based on the polarity of each compound. Among the 5 layers, the chloroform layer had the greatest inhibitory effect on LPS-stimulated nitric oxide production and inducible nitric oxide synthase mRNA expression in Raw 264.7 cells. However, the levels of prostaglandin E2 and cyclooxygease 2 were not altered. On the other hand, treatment of cells with the chloroform layer of A. arguta before LPS stimulation also reduced the mRNA expression of proinflammatory cytokines including tumor necrosis factor α and interleukin 1β. Nuclear translocation of NF-κB p50 and p65 subunits induced by LPS was also inhibited by treatment with the chloroform layer of A. arguta. This was accompanied with the reduced phosphorylation of mitogen-activated protein kinases including extracellular signal-regulated protein kinase 1/2, c-Jun N-terminal protein kinase, and p38. Taken together, these results suggest that chloroform layer of A. arguta exerted antiinflammatory effects by the inhibition of mitogen-activated protein kinase phosphorylation and nuclear translocation of NF-κB.     

花色苷对脂多糖诱导巨噬细胞炎症反应的影响

目的探讨花色苷对脂多糖(LPS)所诱导的THP-1样巨噬细胞中诱导性一氧化氮合酶(iNOS),环氧合酶-2(COX-2)及其产物一氧化氮(NO),前列腺素E2(PGE2)表达的影响。方法THP-1单核细胞用终浓度为167nmol/L的佛波酯(PMA)刺激48h诱导成THP-1样巨噬细胞后,分别用1、10、50、100μmol/L的花色苷标准品矢车菊素-3-葡萄糖苷(Cy-3-g)或芍药素-3-葡萄糖苷(Pn-3-g)孵育细胞2h,再用1μg/mlLPS处理细胞12h,用半定量RT-PCR法分别检测iNOS、COX-2的基因表达量,用Western blotting检测iNOS、COX-2的蛋白表达量,分别用Griess法和碱性磷酸酶法(EIA)检测THP-1样巨噬细胞培养基中NO及PGE2的含量。结果花色苷能有效的下调iNOS,COX-2的基因及蛋白水平,且其所合成的炎性介质NO和PGE2也显著降低。结论花色苷能够降低LPS所诱导的THP-1样巨噬细胞中iNOS,COX-2及其产物NO,PGE2的表达水平,提示其可能对于治疗各种炎症相关性疾病如动脉粥样硬化等有着重要的意义。     

Activated macrophages increase the susceptibility of rat hepatocytes to ethanol-induced oxidative stress: conflicting effects of nitric oxide

The aim of this study was to examine how macrophages could act on ethanol-induced oxidative stress in rat hepatocytes during inflammatory conditions, well-known to induce nitric oxide (NO) synthase. For this purpose, RAW 264.7 macrophages were added to primary rat hepatocyte cultures. Co-cultures were then supplemented with lipopolysaccharide (LPS) and interferon gamma (IFN) for 18 h, in order to induce NO synthase before the addition of 50 mM ethanol. In cultures of hepatocytes alone, the addition of LPS and IFN protected from ethanol-induced oxidative stress. It has been shown previously that NO generated in hepatocytes was responsible for this effect. When macrophages were added to primary rat hepatocyte cultures supplemented with LPS and IFN, protection provided by NO against ethanol-induced oxidative stress in hepatocytes ceased. Using a pretreatment of macrophages with N(g)-monomethyl-l-arginine, a NO synthase inhibitor, it was concluded that NO generated by macrophages was responsible for macrophage toxicity. Taken together, our observations suggest that NO biosynthesis in hepatocytes protects them from ethanol-induced oxidative stress, whereas NO production in macrophages deprives hepatocytes of this NO protection.     

Inhibition of nitric oxide in LPS-stimulated macrophages of young and senescent mice by δ-tocotrienol and quercetin

Background

Changes in immune function believed to contribute to a variety of age-related diseases have been associated with increased production of nitric oxide (NO). We have recently reported that proteasome inhibitors (dexamethasone, mevinolin, quercetin, δ-tocotrienol, and riboflavin) can inhibit lipopolysaccharide (LPS)-induced NO production in vitro by RAW 264.7 cells and by thioglycolate-elicited peritoneal macrophages derived from four strains of mice (C57BL/6, BALB/c, LMP7/MECL-1^-/- and PPAR-α^-/- knockout mice). The present study was carried out in order to further explore the potential effects of diet supplementation with naturally-occurring inhibitors (δ-tocotrienol and quercetin) on LPS-stimulated production of NO, TNF-α, and other pro-inflammatory cytokines involved in the ageing process. Young (4-week-old) and senescent mice (42-week old) were fed control diet with or without quercetin (100 ppm), δ-tocotrienol (100 ppm), or dexamethasone (10 ppm; included as positive control for suppression of inflammation) for 4 weeks. At the end of feeding period, thioglycolate-elicited peritoneal macrophages were collected, stimulated with LPS, LPS plus interferon-β (IFN-β), or LPS plus interferon-γ (IFN-γ), and inflammatory responses assessed as measured by production of NO and TNF-α, mRNA reduction for TNF-α, and iNOS genes, and microarray analysis.

Results

Thioglycolate-elicited peritoneal macrophages prepared after four weeks of feeding, and then challenged with LPS (10 ng or 100 ng) resulted in increases of 55% and 73%, respectively in the production of NO of 46-week-old compared to 8-week-old mice fed control diet alone (respective control groups), without affecting the secretion of TNF-α among these two groups. However, macrophages obtained after feeding with quercetin, δ-tocotrienol, and dexamethasone significantly inhibited (30% to 60%; P < 0.02) the LPS-stimulated NO production, compared to respective control groups. There was a 2-fold increase in the production of NO, when LPS-stimulated macrophages of quercetin, δ-tocotrienol, or dexamethasone were also treated with IFN-β or IFN-γ compared to respective control groups. We also demonstrated that NO levels and iNOS mRNA expression levels were significantly higher in LPS-stimulated macrophages from senescent (0.69 vs 0.41; P < 0.05), compared to young mice. In contrast, age did not appear to impact levels of TNF-α protein or mRNA expression levels (0.38 vs 0.35) in LPS-stimulated macrophages. The histological analyses of livers of control groups showed lesions of peliosis and microvesicular steatosis, and treated groups showed Councilman body, and small or large lymphoplasmacytic clusters.

Conclusions

The present results demonstrated that quercetin and δ-tocotrienols inhibit the LPS-induced NO production in vivo. The microarray DNA analyses, followed by pathway analyses indicated that quercetin or δ-tocotrienol inhibit several LPS-induced expression of several ageing and pro-inflammatory genes (IL-1β, IL-1α, IL-6, TNF-α, IL-12, iNOS, VCAM1, ICAM1, COX2, IL-1RA, TRAF1 and CD40). The NF-κB pathway regulates the production of NO and inhibits the pro-inflammatory cytokines involved in normal and ageing process. These ex vivo results confirmed the earlier in vitro findings. The present findings of inhibition of NO production by quercetin and δ-tocotrienol may be of clinical significance treating several inflammatory diseases, including ageing process.     

Appropriate dose of parenteral arginine enhances immunity of peripheral blood cells and splenocytes in rats with subacute peritonitis

Background: Arginine deficiency and chronic inflammation may cause immune dysfunction. The authors previously showed that a pharmacological dose of parenteral arginine facilitates ornithine rather than nitric oxide production in subacute peritonitis. Herein, they investigated the effects of different doses of parenteral arginine supplementation on immunocytic subpopulation distribution and function. Materials: Male Wistar rats that underwent cecal punctures for induction of subacute peritonitis were infused with conventional parenteral nutrition solution (1.61% of total calories as arginine) or solutions supplemented with low-, medium-, or high-dose arginine (2.85%, 4.08%, and 6.54% of total calories, respectively) for 7 days. Distributions of T cells, B cells, and monocytes/macrophages and cytokine productions of peripheral blood lymphocytes (PBLs) and splenocytes were analyzed. Results: There were no significant differences in circulating white blood cell numbers and serum tumor necrosis factor (TNF)-α and interferon (IFN)-γ concentrations among groups. Serum nitrate/nitrite (NOx) and interleukin (IL)-2 levels were significantly decreased by arginine in a dose-dependent manner. Animals supplemented with parenteral arginine had significantly decreased productions of concanavalin (Con) A- and lipopolysaccharide (LPS)-stimulated TNF-α in PBLs and splenocytes, spontaneous IL-6 and LPS-stimulated IFN-γ in PBLs, and LPS-stimulated IL-6 in splenocytes. In addition, low-dose arginine significantly increased production of spontaneous IFN-γ in PBLs and splenocytes. High-dose arginine significantly increased spontaneous TNF-α, and Con A stimulated IL-4 and IL-6 in PBLs. Conclusion: Parenteral arginine administration at approximately 4% of total calories may alter PBLs and splenocytic immunity, and >6% of total calories might not be of benefit in rats with subacute peritonitis.     

生脉散对大肠杆菌脂多糖诱导慢性肝衰竭大鼠细胞因子水平的影响

目的探讨生脉散对慢性肝衰竭大鼠大肠杆菌脂多糖（LPS）诱导细胞因子水平的影响。方法采用CCl。混合液腹腔注射复制慢性肝衰竭大鼠模型,观察LPS诱导2h后生脉散后对血清内毒素、细胞因子水平的影响。结果CCl。混合液能明显增加大鼠血清IL-6[（64．50±18．79）pg／mlVS（4．79±0．57）pg／m1]、ICAM-1[（25100．00±5258．85）pg／mlVS（4215．50±942．79）pg／m1]和TNF-α[（17．55±2．39）pg／mlVS（10．92±5．02）pg／m1]水平（P〈0．05）,但对血清LPS水平无明显影响[（0．058±0．007）EU／mlVS（0．040±0．002）EU／ml,P〉0．05];中药生脉散能显著降低CCl。慢性肝衰竭大鼠血清IL-6、ICAM-1和TNF-α水平[分别为（17．20±3．12）pg／ml、（9490．00±2725．78）pg／ml、（3．00±1．00）pg／ml,P〈0．05]。LPS攻击2h后能明显升高CCI。混合液大鼠血清LPS、TNF-α、IL-6、ICAM-1水平[分别为（0．501±0．019）EU／ml、（19750．00±9655．17）pg／ml、（5615．00±490．50）pg／ml、（41000．00±589．88）pg／ml,P〈0．01]。结论在慢性肝衰竭模型中,LPS能增加TNF-α,IL-6和ICAM-1等炎症因子水平,中药生脉散可降低LPS水平,并抑制LPS诱导的炎症因子水平,阻断了炎性介质及LPS本身对机体的损伤。     

Molecular role of ascorbate in enhancement of NO production in activated macrophage-like cell line, J774.1

Ascorbate-enhanced nitric oxide (NO) production in lipopolysaccharide (LPS)- and interferon-gamma (IFN-gamma)-activated macrophage J774.1 cells through the inducible nitric oxide synthase (iNOS) pathway. The iNOS gene was synergistically induced by LPS and IFN-gamma. The inductive mechanism of ascorbate on the iNOS gene was studied by examining the degradation of I kappa B alpha by Western blotting, activation of the nuclear factor kappa B (NF-kappa B) by gel shift assays, and protein levels of interferon regulatory factor 1 (IRF-1) in LPS- and IFN-gamma-activated cells. Ascorbate had no effect on the onset of either I kappa B alpha degradation or the nuclear translocation of NF-kappa B, but it delayed the recovery of I kappa B alpha. The prolonged degradation of I kappa B alpha caused by ascorbate in LPS- and IFN-gamma-activated cells paralleled elevated NF-kappa B binding to DNA, which led to an increase in the iNOS protein level. Ascorbate alone did not induce I kappa B alpha degradation or NF-kappa B activation. Furthermore, ascorbate exerted no effect on the expression of I kappa B alpha and ubiquitin genes in the activated cells. Ascorbate could modulate NF-kappa B DNA binding activity in response to combined LPS and IFN-gamma activation, which increases NO production in activated macrophages.     

Effects of an aqueous extract of cocoa on nitric oxide production of macrophages activated by lipopolysaccharide and interferon-gamma

OBJECTIVE: Macrophages are the primary targets of bacterial lipopolysaccharide (LPS). The effects of cocoa extract on production of nitric oxide (NO) by murine J774.1 macrophages activated by LPS and interferon-gamma (IFN-gamma) were examined. METHODS: Cocoa was suspended in heated water and centrifuged, and the supernatant was then filtered. Nitrite was measured as a quantitative indicator of NO by spectrophotometry. LPS (1.0 mg/mL) and IFN-gamma (100 U/mL) were added to cultured macrophages with 0.05% cocoa extract, 0.25% cocoa extract, or pure water. NO synthesis by macrophages was significantly inhibited by cocoa extract (P < 0.01). RESULTS: The inhibitory effect increased with concentration of the extract (P < 0.01). IFN-gamma (100 U/mL) and, later, LPS (100 microgram/mL) were added, together with 2.0% cocoa or pure water, to cultured macrophages. An inhibitory effect on NO production was observed on addition of only IFN-gamma, but more significant effects were obtained with addition of LPS (P < 0.01) and addition of both was most effective (P < 0.01). CONCLUSIONS: These data suggested that cocoa extract contains a suppressor of NO production in murine macrophages activated by LPS and IFN-gamma. This effect does not appear to be caused merely by neutralization of LPS.     

Chronic ethanol ingestion by mice increases expression of CD80 and CD86 by activated macrophages.

Results from previous studies from our laboratory have shown that T cells obtained from the spleens of C57BL/6 mice that consumed ethanol chronically have increased expression of activation markers and increased second signal-independent production of interferon-gamma (IFN-gamma). We now report that in vitro-activated CD11b(+) splenocytes obtained from C57BL/6 and BALB/c mice that consumed ethanol chronically express increased levels of the T cell co-stimulatory molecules CD80 and CD86. CD11b(+) splenocytes encompass at least two populations: the CD11b(+)Gr.1(-) population, which is primarily monocytes-macrophages, and a smaller CD11b(+)Gr.1(+) population, which is in the myelocytic-monocytic cell series and contains precursors of both macrophages and neutrophils. Evaluation of cultures of purified CD11b(+) cells, obtained from mice that consumed ethanol chronically, incubated overnight, showed increased up-regulation of CD80 and CD86 expression on Gr.1(-) mouse splenic macrophages. Results of functional studies of purified CD11b(+) cells have demonstrated that CD11b(+) cells obtained from C57BL/6 mice that were exposed to ethanol chronically secrete higher levels, in comparison with the levels secreted by CD11b(+) cells obtained from control animals, of nitric oxide and several proinflammatory cytokines after stimulation by the oligodeoxynucleotide (ODN) CpG 1826. These findings indicate that CD11b(+) splenocytes are in some way sensitized to activating stimuli by chronic ethanol exposure in vivo. Such cells may contribute to systemic immunodysregulation, including T-cell activation, by providing abnormal second signals to T cells, or through excessive release of cytokines, such as interleukin (IL)-6 or IL-12.