Characterization of leukotriene production in vivo and in vitro in resident and elicited peritoneal macrophages in chickens and mice.

Previously, we reported differences in arachidonic acid metabolism in elicited chicken peritoneal macrophages when compared with murine resident and elicited peritoneal macrophages. We now describe leukotriene (LT) production in the same systems, using resident (murine) and inflammatory macrophages (from both species). Inflammatory (4- or 42-g Sephadex-elicited) peritoneal macrophages from chickens lacked the capacity to produce LT in vivo (following opsonized zymosan [OZ] stimulation) or in vitro, in response to A23187. In addition, chicken macrophages were unable to metabolize exogenously added LTC4 or LTD4 in vitro. In contrast, resident murine peritoneal macrophages produced measurable quantities of LTs (in vivo) within 5 min with an 8-fold increase after 45 min. LTC4 was effectively converted to LTE4 in vivo in a time-dependent manner (65% LTC4/35% LTE4 after 5 min stimulation with OZ and 6% LTC4/94% LTE4 after 60 min stimulation), but no in vitro. The lack of LTC4 metabolism to LTE4 in vitro could not be explained by cell-cell interaction between adherent and nonadherent cells. LTD4 was not detected under any experimental condition. Murine peritoneal cells incubated with LTD4 (with or without agonist) produced LTE4 in a time-dependent fashion. Addition of L-cysteine (a dipeptidase inhibitor) did not explain the lack of detectable levels of LTD4 following intraperitoneal stimulation with OZ. These results suggest that elicited chicken peritoneal macrophages are incapable of producing LTs compared to murine peritoneal macrophages. In addition, these studies fail to explain the different product profiles with in vivo stimulation of murine peritoneal macrophages as compared to in vitro stimulation.

Dietary n - 3 polyunsaturated fatty acids modify Syrian hamster platelet and macrophage phospholipid fatty acyl composition and eicosanoid synthesis: a controlled study.

The aim of this study was to determine the effects of varying intakes of dietary n - 3 polyunsaturated fatty acids (PUFA) on the fatty acyl composition and arachidonic acid metabolite synthesis of platelets and macrophages in Syrian hamsters consuming diets that were strictly controlled for n - 6 PUFA content. Animals consumed highly controlled diets which were not supplemented with n - 3 PUFA (control) or supplemented with 0.4%, 0.8% or 2% (w/w) n - 3 fatty acids. The content of n - 3 PUFA in cellular phospholipids increased progressively with the intake of n - 3 PUFA, while n - 6 PUFA, including arachidonic acid, decreased despite the constant intake of 18:2(n - 6); this latter effect was more substantial in macrophages than in platelets. The synthesis by stimulated macrophages of prostaglandin E2, 6-keto-prostaglandin F1 alpha, thromboxane B2 and 11- and 15-hydroxyeicosatetraenoic acids decreased with the intake of 0.8% n - 3 PUFA to 30-50% of the control values. Little effect of diets on platelet aggregation and eicosanoid synthesis was observed reflecting the limited effect on platelet arachidonic acid content. The synthesis of 12-hydroxyeicosapentaenoic acid by stimulated platelets increased with n - 3 PUFA consumption in a dose-dependent fashion. Circulating triacylglycerols and HDL-cholesterol were decreased only in animals consuming 2% n - 3 PUFA. The strict control of n - 6 PUFA intake allows the determination of the effects of n - 3 PUFA intake on the measured parameters without confounding effects of other dietary lipids.

Effects of n-3 polyunsaturated fatty acids on the reacylation of arachidonic acid in peritoneal macrophages.

Mouse peritoneal macrophages were prelabelled with [3H]AA and activated ex vivo with phorbol myristate acetate (PMA) in the presence or absence of added n-3 polyunsaturated fatty acids (n-3 PUFA). The n-3 PUFA did not affect the release of [3H]AA from stimulated macrophages. Added AA, enhanced, in a dose-dependent manner, the amount of free [3H]AA in the medium after activating the cells with PMA. Both n-3 PUFA and AA were actively acylated into the lipids of PMA activated macrophages, but the n-3 PUFA did not compete with reacylation of AA. In unstimulated cells, in the absence of PMA, n-3 PUFA effectively competed with AA for acylation into membrane phospholipids. These studies suggest that distinct pools of AA exist in macrophages.

Dietary level of maize oil affects growth and lipid composition of Walker 256 carcinosarcoma.

Walker 256 carcinosarcoma cells (1 x 10(4)) were injected into the right thigh muscle of Sprague-Dawley rats (125 g) consuming isoenergetic (200 g fat/kg) diets containing 20, 100 and 200 g maize oil/kg and 180, 100 or 0 g hydrogenated lard/kg respectively. Ten rats from each dietary regimen were killed every 4th day. Tumours grew rapidly from day 0 to day 8 post-transplant regardless of dietary regimen. However, after 8 d more tumours regressed and there were fewer deaths in animals fed on 200 g maize oil/kg compared with animals fed on 20 or 100 g maize oil/kg. Linoleic acid (LA) levels were higher in phospholipids (PL) of growing tumours than in regressing tumours whereas arachidonic acid levels in PL were lower in growing tumours indicating a possible alteration in the desaturation and elongation of LA. Serum prostaglandin E2 levels were slightly lower in rats with regressing tumours than in rats with growing tumours.

Synthesis of arachidonic acid metabolites by Syrian hamster platelets and peritoneal cells.

In this study, the metabolism of arachidonic acid by hamster platelets and peritoneal macrophages was assessed. Peritoneal macrophages stimulated in vitro with the calcium ionophore A23187 or stimulated in vivo by intraperitoneal injections of opsonized zymosan produced prostaglandin E2, thromboxane B2 (TxB2) and 6-keto-prostaglandin F1 alpha, as determined by radioimmunoassays. Leukotriene B4 (LTB4), and 11- and 15-hydroxyeicosatetraenoic acids (HETE), which were identified by reverse-phase high-performance liquid chromatography coupled with diode array detection, were produced by peritoneal cells stimulated in vitro with A23187 but were not found in the peritoneal exudate following in vivo stimulation with opsonized zymosan. Synthesis of 11- and 15-HETE, but not LTB4, was inhibited by 1 microM indomethacin but not by 10 microM nordihydroguaiaretic acid, which did inhibit LTB4 synthesis. Washed hamster platelets were prepared and shown to synthesize TxB2, 12-HETE and 12-hydroxyheptadecatrienoic acid following stimulation with thrombin. This paper is the first to report on eicosanoid metabolism in tissues related to atherosclerosis, thrombosis and inflammation in hamsters.

Dietary arachidonate enhances tissue arachidonate levels and eicosanoid production in Syrian hamsters.

Eicosanoids are oxidative derivatives of arachidonic acid. When produced in excess many of them are proinflammatory agents. This study investigates whether dietary arachidonic acid enhances arachidonic acid phospholipid content of various tissues and whether this enrichment increases eicosanoid production. Male Syrian hamsters were divided into four groups and fed diets supplemented with ethyl esters of oleic acid, linoleic acid, arachidonic acid or eicosapentaenoic acid. Differences in the composition of the phospholipid fatty acids were monitored in liver, lung, heart, spleen, kidney, testes, macrophages and platelets. In all tissues analyzed, the phospholipid content of arachidonic acid was significantly higher in the arachidonic dietary group compared with all other dietary groups (average > 50% higher). In contrast, increasing dietary linoleic acid by 50% had little effect on altering tissue arachidonic acid levels. Following in vitro stimulation, macrophages and platelets from animals maintained on arachidonic acid produced, in general, the highest levels of eicosanoids compared with cells from animals fed the other diets. Significant differences were observed in prostaglandin E2 (macrophages) and thromboxane B2 (platelets) formation when compared with the oleic acid and eicosapentaenoic acid dietary groups. The data demonstrate that including low to moderate levels of arachidonic acid in the diet increases macrophage and platelet arachidonic acid levels and may augment eicosanoid production.

Effects of polyunsaturated fatty acids on the efficacy of antineoplastic agents toward L5178Y lymphoma cells.

Modification of cultured lymphoma cells (L5178Y) with individual unsaturated fatty acids [oleic acid (OA), linoleic acid (LA), alpha-linolenic acid (alpha-LNA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA)] influenced cell growth and the responses of the cells to the chemotherapeutic agents doxorubicin (DRN), dexamethasone (DEX) and mitomycin-C (MTC). Cell proliferation generally decreased following modification with highly unsaturated fatty acids (> 10 microM). The effects of drugs on growth varied with the type of fatty acid. Preincubation with alpha-LNA enhanced survival of L5178Y cells exposed to DRN. Modification with AA, EPA or DHA (> 10 microM) reduced cell proliferation, particularly when cells were subsequently exposed to 50 or 100 nM DRN. There was no consistent relationship between fatty acid chain length, degree of unsaturation, and survival of cells when exposed to DEX or MTC. The data showed that modification of cultured L5178Y cells with highly unsaturated fatty acids, particularly DHA, enhances the toxic action of chemotherapeutic agents.

Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine.

The "French paradox" (apparent compatibility of a high fat diet with a low incidence of coronary atherosclerosis) has been attributed to the regular drinking of red wine. However, the alcohol content of wine may not be the sole explanation for this protection. Red wine also contains phenolic compounds, and the antioxidant properties of these may have an important role. In in-vitro studies with phenolic substances in red wine and normal human low-density lipoprotein (LDL) we found that red wine inhibits the copper-catalysed oxidation of LDL. Wine diluted 1000-fold containing 10 mumol/L total phenolics inhibited LDL oxidation significantly more than alpha-tocopherol. Our findings show that the non-alcoholic components of red wine have potent antioxidant properties toward oxidation of human LDL.

Dietary n-3 fatty acids alter murine peritoneal macrophage cytotoxicity.

Male CD-1 mice were fed diets containing 10 wt% fat composed of a constant amount of n-6 polyunsaturated fatty acids (PUFA; safflower oil, 1.5 wt%) and increasing levels of n-3 PUFA (up to 1.5 wt%); the remaining fat was a 50:50 mixture of tripalmitin and triolein. Four days prior to sacrifice, the animals were injected intraperitoneally with sterile thioglycollate. Peritoneal macrophages were analyzed for phospholipid fatty acids, prostaglandin E2 (PGE2) synthesis and cytotoxicity towards L929 target cells. Increasing dietary n-3 PUFA increased macrophage phospholipid n-3 PUFA from 4.4 to 11.0 mol% with a concomitant decline in n-6 PUFA from 15.5 to 10.9 mol%, and PGE2 synthesis declined from 2.6 to 1.5 pmol/million cells in thioglycollate-elicited macrophages. Macrophage cytotoxicity toward target L929 cells declined by approximately 50% with increasing n-3 PUFA concentration.

Increasing the dietary (n-3) to (n-6) polyunsaturated fatty acid ratio increases tumor necrosis factor production by murine resident peritoneal macrophages without an effect on elicited peritoneal macrophages.

Tumor necrosis factor (TNF), prostaglandin (PG) E2 and 6-keto-PGF1 alpha production by murine peritoneal macrophages was monitored following in vitro stimulation with lipopolysaccharide. Macrophages were obtained from mice fed diets containing increasing ratios of (n-3) to (n-6) fatty acids by addition of (n-3) polyunsaturated fatty acids (PUFA) to the (n-6) fatty acids in the diet, or by substituting (n-3) PUFA for the (n-6) fatty acids in the diet. Increasing the dietary (n-3) to (n-6) fatty acid ratio from 0 to 1 increased both cell-associated and secreted TNF production by resident peritoneal macrophages but did not affect TNF production by macrophages elicited with Complete Freund's Adjuvant (CFA). With increasing dietary (n-3): (n-6) ratio there was a decrease in the prostaglandin production by resident peritoneal macrophages, which may partly explain the increased TNF production. The CFA-elicited macrophages produced less prostaglandin than the resident macrophages, and the lower prostaglandin production may partly explain the lack of effect of dietary (n-3) PUFA on TNF production by CFA-elicited macrophages. Increasing the TNF production by resident macrophages with dietary (n-3) PUFA may be beneficial in enhancing antitumor actions and antipathogenicity; by not increasing the high TNF production of inflammatory macrophages, (n-3) PUFA may protect against undesirable systemic inflammatory effects of overproduction.

Kinetics of tumour necrosis factor and prostaglandin production by murine resident peritoneal macrophages as affected by dietary n-3 polyunsaturated fatty acids.

Cell-associated and secreted tumour necrosis factor (TNF), prostaglandin (PG) E2, and 6-keto PGF1 alpha were monitored at various times following in vitro stimulation of resident peritoneal macrophages with lipopolysaccharide (LPS). Macrophages were obtained from mice maintained on diets containing 1.5 wt% n-3 polyunsaturated fatty acids (PUFA)+ 1.5 wt% n-6 fatty acids; 1.5 wt% n-6 fatty acids; or 3 wt% n-6 fatty acids, for 4 weeks. Cell-associated TNF increased transiently in the resident peritoneal macrophages from mice consuming all diets and decreased after TNF secretion had reached maximum and plateaued. Macrophages from mice consuming the n-3 PUFA contained more cell-associated TNF and secreted more TNF than macrophages from mice consuming diets containing n-6 fatty acids only, at all time-points studied. Macrophages from mice consuming the n-3 PUFA showed an earlier increase in cell-associated and secreted TNF compared with macrophages from mice consuming n-6 fatty acids only. Kinetics of maximum TNF production was not affected by the diets and dietary n-3 PUFA did not cause a prolonged increase in TNF secretion. Macrophages from mice consuming the n-3 PUFA produced less PG than macrophages from mice consuming the n-6 fatty acids only. PG secretion increased following appearance of cell-associated TNF but when PG had accumulated in the medium there was no further increase in TNF production.

Effect of different dietary triglycerides on liver fatty acids and prostaglandin synthesis by mouse peritoneal cells.

The effect of dietary triglycerides varying in fatty acid composition on the tissue fatty acids and prostaglandin synthesis was studied in mice. The dietary fats were medium-chain triglycerides (rich in C8:0 and C10:0), structured lipids (rich in 12:0), high oleic sunflower oil (rich in 18:1), corn oil (rich in n-6 polyunsaturated fatty acids), and menhaden oil (rich in n-3 polyunsaturated fatty acids) fed at 5% by weight in refined diets. The medium chain fatty acids C8 to C12 from medium-chain triglycerides and structured lipids did not accumulate in liver phospholipids. However, long-chain fatty acids from the dietary fats were incorporated into liver lipids, with n-3 polyunsaturated fatty acids replacing arachidonic acid. The synthesis of 6-keto-prostaglandin F1 alpha and prostaglandin E2 by peritoneal cells in response to intraperitoneal injection of zymosan decreased as the arachidonic acid levels were decreased. When the same dietary fats were added to the refined, fat-free diets, at 7.5 wt% levels, together with 2.5 wt% of safflower oil to provide essential fatty acids, only the long-chain fatty acids from the dietary fats were incorporated into the liver lipids. The arachidonic acid in liver lipids was enhanced after supplementation of diets with safflower oil. However, the reduction in prostaglandin synthesis by peritoneal cells in response to intraperitoneal injection of zymosan was similar to that observed when 5% fat was fed. The data suggest that dietary fats of defined composition, with or without added essential fatty acids, may be useful as alternate fat sources in parenteral nutrition in reducing inflammatory responses mediated via prostaglandins.

Evidence for mechanisms of the hypotriglyceridemic effect of n-3 polyunsaturated fatty acids.

Ingestion of modest amounts of n-3 polyunsaturated fatty acids (PUFA) (2.8% w/w) decreased plasma triglyceride levels in Syrian hamsters by 49%. This was associated with a 45% increase in hepatic carnitine palmitoyl transferase activity. Significantly, at this low level of n-3 PUFA intake, hepatic peroxisomal oxidation measured as CN- insensitive palmitoyl-CoA dependent NAD reduction was unaffected. Consumption of increasing amounts of dietary n-3 PUFA up to 2% (w/w) in hamster diets containing 15% fat, gradually decreased plasma triglycerides to 56% of the control levels. The diet induced changes in plasma triglyceride levels were highly correlated (r = -0.97, P less than 0.01) with changes in hepatic carnitine palmitoyl transferase activity. A gradual decrease up to 59% in hepatic phosphatidate phosphohydrolase activity with n-3 fatty acid consumption was also observed. The hypotriglyceridemic effects of moderate intakes of n-3 fatty acids are, therefore, associated with changes in key enzymes in hepatic triglyceride synthesis and mitochondrial oxidation, but not peroxisomal oxidation.

Dependence on dietary cholesterol for n-3 polyunsaturated fatty acid-induced changes in plasma cholesterol in the Syrian hamster.

Male Syrian hamsters consumed diets containing incremental increases in dietary n-3 fatty acids from fish oil with either low (0.015% w/w) or moderate (0.1% w/w) dietary cholesterol content. Animals consuming diets containing moderate cholesterol, but not animals consuming diets containing low cholesterol, had increased plasma very low (VLDL)- and low density lipoprotein (LDL)-cholesterol levels with increasing fish oil consumption. The plasma concentration of high density lipoprotein (HDL)-cholesterol decreased by 43 and 32% with the consumption of the highest fish oil diets in the low and moderate dietary cholesterol groups, respectively. Hepatic LDL-receptor binding activity did not change with the consumption of low cholesterol diets, but gradually decreased with fish oil consumption in animals consuming the moderate cholesterol diets. Hepatic LDL-receptor binding and plasma LDL-cholesterol levels of the different dietary fish oil groups were highly correlated (r = -0.91). Fish oil consumption also caused an increase in hepatic free cholesterol but a decreased cholesteryl ester content. Therefore, in the Syrian hamster, the consumption of n-3 fatty acids increases LDL-cholesterol levels which can be partially explained by decreased hepatic LDL-receptor binding and this response to dietary n-3 fatty acids is dependent on the dietary cholesterol content. However, the effects of dietary n-3 fatty acids on HDL-cholesterol are independent of dietary cholesterol content.

Review of the Streptomyces lividans/vector pIJ702 system for gene cloning.

Interest in the biology of the Streptomyces and application of these soil bacteria to production of commercial antibiotics and enzymes has stimulated the development of efficient cloning techniques and a variety of streptomycete plasmid and phage vectors. Streptomyces lividans is routinely employed as a host for gene cloning, largely because this species recognizes a large number of promoters and appears to lack a restriction system. Vector pIJ702 was constructed from a variant of a larger autonomous plasmid and is often used as a cloning vehicle in conjunction with S. lividans. The host range of vector pIJ702 extends beyond Streptomyces spp., and its high copy number has been exploited for the overproduction of cloned gene products. This combination of host and vector has been used successfully to investigate antibiotic biosynthesis, gene structure and expression, and to map various Streptomyces mutants.

Dietary arachidonic and linoleic acids: comparative effects on tissue lipids.

The effects of preformed dietary arachidonic acid (AA, 20:4n-6) on murine phospholipid fatty acid composition in tissues capable (liver) and incapable (peritoneal exudate cells, PEC) of desaturating and elongating linoleic acid (LA, 18:2n-6) to AA were investigated. The results were compared with those obtained on matched animals on LA diets by either substituting or supplementing dietary LA with AA. Modest amounts of AA ethyl ester (0.5 wt%) included in the diet significantly increased tissue phospholipid AA levels by 39% and 57% in the liver and in PEC, respectively. The changes were further enhanced when dietary LA and AA intakes were equivalent, i.e., 57% and 68% in liver and PEC, respectively. This enrichment was observed in all phospholipid classes analyzed, with the greatest impact on phosphatidylcholine. In addition, the doubling of dietary LA had little effect on tissue phospholipid AA levels. The data suggest that while the level of n-6 PUFA may have an important effect on tissue fatty acid composition, the type of n-6 PUFA in the diet could be of greater significance.

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.

Effect of increasing the dietary (n-3) to (n-6) polyunsaturated fatty acid ratio on murine liver and peritoneal cell fatty acids and eicosanoid formation.

An incremental increase in the dietary (n-3):(n-6) polyunsaturated fatty acid (PUFA) ratio from 0 to 1.93 in diets containing 15% fat (wt/wt) decreased the total (n-6) PUFA content of phospholipids of the liver and peritoneal cells (macrophage) in mice from 43.1 and 33.6 mol/100 mol to 16.0 and 12.3 mol/100 mol with a concomitant increase of 27.6 and 16.1 mol/100 mol in (n-3) PUFA, respectively. Consumption of (n-3) PUFA increased hepatic (n-3) PUFA levels without changing total PUFA (46.35 vs. 46.87 mol/100 mol), whereas macrophage PUFA levels were decreased. The synthesis of sulfidopeptide leukotrienes (SP-LT) (LTC4 and LTE4) was progressively reduced by increasing dietary (n-3) PUFA, i.e., there was a reduction of 76% in mice fed a diet containing a (n-3):(n-6) PUFA ratio of 1.93 compared with the control diet. The 5-series SP-LT (LTC5 and LTF5) were produced in all animals consuming (n-3) PUFA and accounted for 62% of all SP-LT synthesized in mice fed the diet containing a 1.93 (n-3):(n-6) PUFA ratio. Synthesis of 6-keto-prostaglandin F1 alpha decreased 81% in mice fed a diet containing a (n-3):(n-6) PUFA ratio of 1.93 whereas prostaglandin E2 synthesis decreased 44% in mice fed diets with (n-3):(n-6) ratios ranging from 0.41 to 1.93.