Tumor necrosis factor production by murine resident peritoneal macrophages is enhanced by dietary n-3 polyunsaturated fatty acids

Tumor necrosis factor (TNF) is a macrophage derived peptide that has an antitumor action and modulates immune and inflammatory reactions. Dietary fatty acids may modulate TNF production as dietary n-3 polyunsaturated fatty acids suppress human monocyte TNF production, but enhance its secretion by murine peritoneal macrophages. Mice were maintained for 5 weeks on diets containing different amounts of n-3 and n-6 fatty acids. TNF, PGE2 and 6-keto PGF1 alpha production was monitored following in vitro stimulation of resident peritoneal macrophages with lipopolysaccharide. Macrophages from mice fed the high n-3 diet produced 8-fold more TNF and half the PGE2 produced by macrophages from mice on the other diets. Indomethacin caused an increase in the TNF production by macrophages from mice on all diets but macrophages from mice on the high n-3 diet produced more TNF than macrophages from mice on the other diets. Exogenous PGE2 (100 nM) greatly decreased TNF production by macrophages from mice on all diets, but macrophages from mice on the high n-3 diet secreted 70% more TNF than macrophages from mice fed the other diets, indicating that PGE2 is only partly responsible for the effects observed. The results show that feeding n-3 polyunsaturated fatty acids may cause enhanced TNF production by resident peritoneal macrophages and that PGE2 is partly responsible for the effect.     

Eicosadienoic acid differentially modulates production of pro-inflammatory modulators in murine macrophages

Eicosadienoic acid (Δ11,14-20:2; EDA) is a rare, naturally occurring n-6 polyunsaturated fatty acid (PUFA) found mainly in animal tissues. EDA is elongated from linoleic acid (LA), and can also be metabolized to dihomo-γ-linolenic acid (DGLA), arachidonic acid (AA), and sciadonic acid (Δ5,11,14-20:3; SCA). Although, the metabolism of EDA has been extensively studied, there are few reports regarding how EDA might affect inflammatory processes. The objective of this study was to determine the effect of EDA on the n-6 PUFA composition and inflammatory response of murine RAW264.7 macrophages to lipopolysaccharide (LPS). EDA was taken up rapidly by macrophages and metabolized to SCA, and the percentages of both fatty acids increased in cellular phospholipids in a dose-dependent manner. The incorporation of EDA into macrophage lipids increased the proportions of LA, DGLA, and AA as well, and reduced the proportion of total monounsaturated fatty acids. When LPS were applied to the macrophages, EDA decreased the production of nitric oxide (NO), and increased that of prostaglandin E(2) (PGE(2)) and tumor necrotic factor-α. The modulation of NO and PGE(2) was due, in part, to the modified expression of inducible nitric oxide synthase and type II cyclooxygenase. The differential effects of EDA on pro-inflammatory mediators might attribute to the negative feedback mechanism associated with prolonged inflammation. Furthermore, EDA was a weaker pro-inflammatory agent than LA, and not as anti-inflammatory as SCA. This study shows that EDA can modulate the metabolism of PUFA and alter the responsiveness of macrophages to inflammatory stimulation.     

Citral and eugenol modulate DNA damage and pro-inflammatory mediator genes in murine peritoneal macrophages

Citral and eugenol have been broadly studied because of their anti-inflammatory, antioxidant and antiparasitic potentials. In this study, the effects of citral (25, 50 and 100 µg/mL) and eugenol (0.31, 0.62, 1.24 and 2.48 µg/mL) on the expression (RT-PCR) of the pro-inflammatory mediator genes NF-κB1, COX-2 and TNF-α were evaluated in mouse peritoneal macrophages with or without activation by a bacterial lipopolysaccharide (LPS). Additionally, the genotoxic potentials of two compounds and their capacities to modulate the DNA damage induced by doxorubicin (DXR) were investigated using the comet assay. The data revealed that neither citral nor eugenol changed COX-2, NF-κB1 or TNF-α expression in resting macrophages. However, in LPS-activated cells, citral induced the hypoexpression of COX-2 (100 µg/mL) and TNF-α (50 and 100 µg/mL). Hypoexpression of TNF-α was also detected after cellular exposure to eugenol at the highest concentration (2.48 µg/mL). Both compounds exhibited genotoxic potential (citral at 50 and 100 µg/mL and eugenol at all concentrations) but also showed chemopreventive effects, in various treatment protocols. Both citral and eugenol might modulate inflammatory processes and DXR-induced DNA damage, but the use of these compounds must be viewed with caution because they are also able to induce primary DNA lesions.     

Activation of murine macrophages by hydrogen peroxide

Hydrogen peroxide at concentrations from 0.1 to 20 μM enhances phagocytosis and oxidative burst of murine peritoneal macrophages. The activation of these macrophage functions is paralled by prolonged hyperpolarization and a transient increase in cytoplasmic free calcium concentration. All the effects are dose- and time-dependent. The results obtained for H₂O₂ are compared with those for a natural activator, peptide N-formyl-methionyl-leucly-phenylalanine. The data demonstrate the ability of small doses of hydrogen peroxide to stimulate macrophages through the intracellular mechanisms of ion transduction.     

Activation by fatty acids of the production of active oxygen species by tobacco cells

Among the different transduction steps leading from hypoosmotic stress to oxidative burst in suspension-cultured tobacco (Nicotiana tabacum cv Xanthi) cells, phospholipase activation was evidenced. Using thin layer chromatography and phospholipase inhibitors the involved lipase was strongly suggested to be a phospholipase A₂ (EC 3.1.1.4). Fatty acids like arachidonate and linolenate stimulated the oxidative response and prevented its inhibition by a phospholipase inhibitor, confirming the physiological relevance of the phospholipase action. A production of active oxygen species by plasma membrane vesicles was demonstrated, using two different probes. The producing system characterized in vitro was NADPH-dependent, strongly depressed by iodonium diphenyl and activated by fatty acids like the oxidative response assayed in vivo. Several other anionic amphiphiles like SDS were able to mimic the activation of the oxidative response by fatty acids, both in vivo and in vitro, suggesting that negative charges may be involved in the action mode of fatty acids. Inversely, a cationic detergent was an efficient inhibitor of the hypoosmotically induced oxidative burst and the inhibition was fully reversed by SDS. The possible identification of the active oxygen synthase involved in hypoosmotic signalling with a plasma membrane-located NADPH oxidase is discussed.     

The effect of dietary n-3 and n-6 fatty acids on tumor necrosis factor-alpha production and leucine aminopeptidase levels in rat peritoneal macrophages

The purpose of this study was to determine the effect of dietary n-3 and n-6 fatty acids on tumor necrosis factor-alpha (TNF-alpha) production and macrophage (MO) activation state. Rats were fed diets containing 12.5% linseed oil (LO) or corn oil (CO) that are high in n-3 and n-6 fatty acids respectively. The LO diet resulted in a significant increase in basal and endotoxin (LPS)-induced levels of TNF-alpha from resident MO cultured in vitro. There was no difference between the diets in LPS-induced TNF-alpha production by complete Freund's adjuvant (CFA) elicited macrophages. Variable responses were also observed between LO and CO MO in response to prostaglandin E2, indomethacin (INDO), and the prostaglandin E receptor antagonist SC-19220. This may indicate differences in signal transducing secondary messengers due to different activation states, receptor expression or ligand binding. Fluorescence due to leucine aminopeptidase (LAP) staining was determined by flow cytometry. Resident LO MO had a 15% increase in LAP fluorescence compared to CO MO. In CFA-elicited MO, the CO MO had a 43% increase in fluorescence compared to LO MO. Resident LO MO increased in LAP fluorescence by 35% to the activated state whereas resident CO MO increased in LAP fluorescence by 93%. The smaller window of activation for the LO MO may explain some of the antiinflammatory properties of dietary n-3 fatty acids.     

Activation of fatty acids by non-glyoxysomal peroxisomes

Ethylene treatment (approx. 20 l ·1^-1 in air for 2 d) of tobacco (Nicotiana tabacum L. cv. Havana 425) plants markedly increases the endo--1,3-glucanase (EC 3.2.1.39) content of leaves. The antigenic form of the enzyme induced is the same one whose production is blocked by treating cultured cells with combinations of auxin (1.1 · 10^-5 M -naphthaleneacetic acid) and cytokinin (1.4 · 10^-6 M kinetin). Evidence is presented that cultured tobacco cells require ethylene for -1,3-glucanase accumulation: i) ethylene treatment increased the accumulation of \-1,3-glucanase in callus tissues >10 d after subculturing and in cell-suspension cultures; ii) callus tissues can produce ethylene; iii) conditions known to inhibit ethylene production (1 mM CoCl₂; 33° C treatment) or ethylene action (approx. 1.6 mmol · 1^-1 norbornadiene in air) inhibited -1,3-glucanase accumulation by callus tissues treated for 4 d following subculturing; and, these inhibitory effects were prevented by exogenous ethylene. Combinations of auxin and cytokinin blocked ethylene-induced accumulation of -1,3-glucanase by cell-suspension cultures. The results favor a model in which ethylene induces results favor a model in which ethylene induces 1,3-glucanase accumulation, and auxin and cytokinin inhibit this induction process.Abbreviations NAA -naphthaleneacetic acid - NDE norbornadiene     

Unsaturated fatty acids stimulate NADPH-dependent superoxide production by cell-free system derived from macrophages

Arachidonic acid (C20:4) and other unsaturated fatty acids are shown to activate superoxide (O₂^?) production in a cell-free system represented by sonically disrupted guinea pig peritoneal macrophages. The reaction requires a heat-sensitive cellular component and NADPH, is enhanced by flavin adenine dinucleotide (FAD), and is not linked to enzymatic oxidation of the fatty acid. C20:4-elicited O₂^? formation is dependent on the cooperation between a subcellular component sedimentable at 48,000g (probably containing the O₂^?-forming enzyme) and a cytosolic factor. This appears to be the first report of O₂^? generation being elicited in a cell-free system derived from unstimulated cells and supports the idea that unesterified unsaturated fatty acids act as second messengers of O₂^? formation in intact phagocytes.     

Suppressive effect of short-chain fatty acids on production of proinflammatory mediators by neutrophils

Short chain fatty acids (SCFAs) are fermentation products of anaerobic bacteria. More than just being an important energy source for intestinal epithelial cells, these compounds are modulators of leukocyte function and potential targets for the development of new drugs. The aim of this study was to evaluate the effects of SCFAs (acetate, propionate and butyrate) on production of nitric oxide (NO) and proinflammatory cytokines [tumor necrosis factor α (TNF-α) and cytokine-induced neutrophil chemoattractant-2 (CINC-2αβ)] by rat neutrophils. The involvement of nuclear factor κB (NF-κB) and histone deacetylase (HDAC) was examined. The effect of butyrate was also investigated in vivo after oral administration of tributyrin (a pro-drug of butyrate). Propionate and butyrate diminished TNF-α, CINC-2αβ and NO production by LPS-stimulated neutrophils. We also observed that these fatty acids inhibit HDAC activity and NF-κB activation, which might be involved in the attenuation of the LPS response. Products of cyclooxygenase and 5-lipoxygenase are not involved in the effects of SCFAs as indicated by the results obtained with the inhibitors of these enzymes. The recruitment of neutrophils to the peritonium after intraperitoneal administration of a glycogen solution (1%) and the ex vivo production of cytokines and NO by neutrophils were attenuated in rats that previously received tributyrin. These results argue that this triglyceride may be effective in the treatment of inflammatory conditions.     

Motion of spin-labeled fatty acids in murine macrophages relation to cellular phagocytic activity

Macrophage membrane fluidity was investigated with respect to cellular phagocytic activity through the use of fatty acid spin labels.Spin-labeled fatty acid derivatives were incorporated into intact mouse peritoneal macrophages by exchange from bovine serum albumin. The electron spin resonance (ESR) spectra of the spin-labeled fatty acids in the macrophages showed a pronounced temperature dependence and a decrease in the hyperfine splittings ($\text{2T}{}_{\mathrm{|}}$) of the spectra as the nitroxide radical was moved away from the polar head group of the fatty acid derivatives.Spin-labeled macrophages underwent a time- and temperature-dependent decay, which was inhibited by preincubating the cells with mercuric chloride, heating at 56 °C, or by fixing them with 0.25 % glutaraldehyde.No correlation between the phagocytic activity of macrophages and membrane freedom of motion could be demonstrated. Treatment of macrophages with anti-macrophage serum or extended in vitro cultivation inhibited cellular phagocytic activity but exerted no effect on the motional freedom of the macrophage membrane. Enrichment of the fatty acid composition of the macrophage membrane with $\text{cis-}$ or $\text{trans-}\text{unsaturated}$ fatty acids had striking effects on cellular phagocytic activity, while no significant changes could be detected in the freedom of motion of incorporated fatty acid spin labels at the degree of specific enrichment achieved here. Thus no correlation between cellular phagocytic activity and lipid motion could be detected.     

Metabolism of glucose, glutamine, long-chain fatty acids and ketone bodies by murine macrophages.

Maximum activities of some key enzymes of metabolism were studied in elicited (inflammatory) macrophages of the mouse and lymph-node lymphocytes of the rat. The activity of hexokinase in the macrophage is very high, as high as that in any other major tissue of the body, and higher than that of phosphorylase or 6-phosphofructokinase, suggesting that glucose is a more important fuel than glycogen and that the pentose phosphate pathway is also important in these cells. The latter suggestion is supported by the high activities of both glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. However, the rate of glucose utilization by 'resting' macrophages incubated in vitro is less than the 10% of the activity of 6-phosphofructokinase: this suggests that the rate of glycolysis is increased dramatically during phagocytosis or increased secretory activity. The macrophages possess higher activities of citrate synthase and oxoglutarate dehydrogenase than do lymphocytes, suggesting that the tricarboxylic acid cycle may be important in energy generation in these cells. The activity of 3-oxoacid CoA-transferase is higher in the macrophage, but that of 3-hydroxybutyrate dehydrogenase is very much lower than those in the lymphocytes. The activity of carnitine palmitoyltransferase is higher in macrophages, suggesting that fatty acids as well as acetoacetate could provide acetyl-CoA as substrate for the tricarboxylic acid cycle. No detectable rate of acetoacetate or 3-hydroxybutyrate utilization was observed during incubation of resting macrophages, but that of oleate was 1.0 nmol/h per mg of protein or about 2.2% of the activity of palmitoyltransferase. The activity of glutaminase is about 4-fold higher in macrophages than in lymphocytes, which suggests that the rate of glutamine utilization could be very high. The rate of utilization of glutamine by resting incubated macrophages was similar to that reported for rat lymphocytes, but was considerably lower than the activity of glutaminase.     

Evaluation of inhibitory activities of plant extracts on production of LPS-stimulated pro-inflammatory mediators in J774 murine macrophages

Whole plant methanolic extracts of 14 traditionally used medicinal herbs were evaluated for their anti-inflammatory activity. Extracts of Grindelia robusta, Salix nigra, Arnica montana, and Quassia amara showed up to 4.5-fold inhibition of nitric oxide (NO) production in the J774 murine macrophage cells challenged with LPS without cytotoxicity. These four selected extracts significantly reduced the protein levels of inducible NO synthase (iNOS) and the cyclooxygenase-2 (COX-2) as observed by Western blot analysis. Culture supernatants from cells treated with these extracts indicated 3–5-fold reduction of tumor necrosis factor-α (TNF-α). However, only G. robusta and Q. amara extracts significantly inhibited (by 50%) IL-1β and IL-12 secretions. Furthermore, all these plant extracts were shown to prevent the LPS-mediated nuclear translocation of nuclear factor-κB (NF-κB). All the above observations indicate the anti-inflammatory potential of these plant extracts.     

5-series peptido-leukotriene synthesis in mouse peritoneal macrophages: modulation by dietary n-3 fatty acids

This study was undertaken to elucidate the metabolic fate of dietary eicosapentaenoic acid (20:5n-3), a major n-3 polyunsaturated fatty acid constituent of fish oil, in the mouse peritoneal macrophage. Mice were fed diets containing menhaden fish oil (FO) or safflower oil (SO). After 3 weeks, resident or responsive peritoneal macrophages were isolated and stimulated with zymosan and A23187, respectively. We demonstrate the novel synthesis of leukotriene C5 (LTC5), derived from 20:5n-3, in addition to LTC4 in both resident and responsive macrophages from FO fed mice. The structures of these peptido-leukotrienes were characterized by their retention times on reverse phase high pressure liquid chromatography, by their u.v. absorbance spectra, and by cross-reactivity with peptido-leukotriene antibody. These results demonstrate for the first time that macrophages are capable of metabolizing dietary 20:5n-3 to LTC5, a pentaene peptido-leukotriene.