Biological response of guinea pig peritoneal macrophages to platelet-activating factor

We investigated the effects of platelet-activating factor (PAF) on guinea pig peritoneal macrophages. Specific and high-affinity binding sites for PAF were detected on guinea pig peritoneal macrophages. Scatchard analysis of PAF binding revealed high affinity binding sites (7.9×10⁴/cell) with a dissociation constant of 2.3×10⁻¹⁰ M. When treated with 10⁻⁹−10⁻⁵ M PAF, guinea pig peritoneal macrophages released hydrogen peroxide into the medium in a time-dependent manner. The release reaction upon stimulation with 10⁻⁵ M PAF reached a plateau within 30 min and the extent of release was twice as high as that when stimulated byN-formyl-L-methionyl-leucyl-L-phenylalanine (fMLP; 2 μM)-treated cells. Neither lysoPAF nor the PAF enantiomer was effective. PAF-induced H₂O₂ release was inhibited specifically by PAF antagonists, suggesting that PAF activated macrophages through binding to specific sites. Lysosomal enzyme (N-acetyl-β-D-glucosaminidase) was released from guinea pig peritoneal macrophages upon treatment with 10⁻⁵ M PAF for 60 min. Guinea pig peritoneal macrophages were treated with PAF for 8 hr and the conditioned medium was examined for cytokines. The medium exhibited cytocidal activity against mouse fibroblast L929 cells [tumor necrosis factor (TNF) activity], and this activity was comparable to that detected after treatment of cells with the bacterial lipopolysaccharide (LPS). Furthermore, the same conditioned medium also showed colony-stimulating factor (CSF) activity. Generation of these cytokines was stereospecific. Our findings suggest that PAF is a unique macrophage activator that potentiates both respiratory burst/lysosomal enzyme release (early-phase response) and monokine production/glucose consumption (late-phase response). Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Stimulation of a neutral triacylglycerol hydrolase from rat heart by phosphatidylethanolamine and lysophosphatidylethanolamine

Triacylglycerol hydrolase activity measured at pH 7.5 in a pH 5.2 precipitate fraction from rat heart was increased two-to three-fold by the presence of phosphatidylethanolamine (PE) or lysophosphatidylethanolamine (LPE). This stimulatory effect also could be obtained in assays with particulate and soluble subcellular fractions and was observed with two different methods of preparing triolein substrate emulsions. Ethanolamine and glycerophosphorylethanolamine had no effect on hydrolase activity, whereas phosphatidylcholine (PC) and acidic phospholipids such as cardiolipin were inhibitory. Palmitic acid, palmityl CoA and palmityl carnitine inhibited PE-stimulated hydrolase activity, but ethyl esters of palmitate had no effect. The preparation of acetone-ether powders resulted in a marked reduction of triacylglycerol hydrolase activity, but PE and LPE now stimulated hydrolase activity by ten-fold or greater, suggesting that these phospholipids may have an obligatory role in modulating triacylglycerol hydrolase activity. Triton X-100 also stimulated hydrolase activity in acetone-ether powders.     

Transfer of arachidonic acid from lymphocytes to macrophages

The incorporation and oxidation of arachidonic acid (AA) by rat lymphocytes (LY), the transfer of AA from LY to rat macrophages (Mϕ) in co-culture, and the subsequent functional impact on Mϕ phagocytosis were investigated. The rate of incorporation of [1-¹⁴C]AA by untreated-LY and TG (thioglycolate treated)-LY (TG-LY) was 158±8 nmol/10¹⁰ LY per h for both untreated-LY and TG-LY. The oxidation of AA was 3.4-fold higher in TG-LY as compared with untreated cells. LY from TG-injected rats had a 2.5-fold increase in the oxidation of palmitic (PA), oleic (OA), and linoleic (LA) acids. After 6 h of incubation, [¹⁴C] from AA was distributed mainly into phospholipids. The rate of incorporation into total lipids was 1071 nmol/10¹⁰ cells in untreated-LY and 636 nmol/10¹⁰ cells in TG-LY. [¹⁴C]AA was transferred from LY to co-cultured Mϕ in substantial amounts (8.7 nmol for untreated and 15 nmol per 10¹⁰ for TG cells). Exogenously added AA, PA, OA, and LA caused a significant reduction of phagocytosis by resident cells. Mϕ co-cultured with AA-preloaded LY showed a significant reduction of the phagocytic capacity (about 40% at 35 μM). LY preloaded with PA, LA, and OA also induced a reduction in phagocytic capacity of co-cultured Mϕ. TG treatment abolished the AA-induced inhibition of phagocytosis in Mϕ co-cultured with TG-LY. Therefore, the transfer of AA between leukocytes is a modulated process and may play an important role in controlling inflammatory and immune response.     

Synthesis of triacylglycerol by lipase in phosphatidylcholine reverse micellar system

Triacylglycerols were synthesized from 1,2-diacylglycerol and fatty acids by lipase entrapped in phosphatidylcholine reverse micelles in n-hexane. In the reaction system without reverse micelles, however, 1,2-diacylglycerol was hydrolyzed into 2-monoacylglycerol and fatty acid, and triacylglycerol was not synthesized. The maximum activity of synthetic reaction was obtained at Wo=10 (Wo=mol water/mol surfactant), which was the water content of this reverse micellar system. Though the optimal pH of theR. delemar lipase reaction is about pH 5.6 in a bulk water system, the enzyme was active for triacylglycerol synthesis at pH’s from 5 to 9 in the reverse micellar system. For the synthesis of triacylglycerols, lauric, myristic, palmitic, stearic, oleic and arachidic acids were effectively used as the fatty acid substrate. 2-Monoacylglycerol was also effective as a substrate of triacylglycerol synthesis. Furthermore, 1,2-diacylglycerol could be replaced by several kinds of aliphatic alcohols as fatty acid acceptors in the reverse micellar system. In this case, those alcohols with chain length more than 4 carbons were effectively used for ester formation.     

Preparation of hydroperoxy and hydroxy derivatives of rat liver phosphatidylcholine and phosphatidylethanolamine

A convenient method for the preparation of hydroperoxy and hydroxy derivatives of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is described. PC and PE obtained from rat liver were oxidized with singlet oxygen by using methylene blue as the photosensitizer, and their hydroperoxides were isolated with the aid of reverse phase liquid chromatography. The hydroxy derivatives were obtained by reducing the hydroperoxides with sodium borohydride. The results of gas chromatography mass spectrometry revealed that hydroxy fatty acid components of the hydroxy derivatives were derived from isomeric hydroperoxides of oleic acid, linoleic acid, arachidonic acid and docosahexanoic acid. Normal phase high performance liquid chromatography did not separate the hydroperoxy and hydroxy derivatives from the respective unoxidized phospholipids, although unoxidized PC and PE were separated from each other. However, the hydroperoxy and hydroxy derivatives could be distinguished from unoxidized phospholipid species on reversed phase thin layer chromatography.     

Synthesis of phosphatidylcholine and phosphatidylethanolamine at different ages in the rat brain in vitro

The de novo synthesis of choline and ethanolamine phosphoglycerides in brain microsomes from 18 month-old male rats was investigated in vitro by using labeled cytidine-5′-diphosphate choline and cytidine-5′-diphosphate ethanolamine as lipid precursors. The rate of synthesis of the two phospholipid classes was found to be noticeably decreased, as compared to that of adult animals. The addition of exogenous diacyl glycerols to microsomes from ageing rat brain brings the rate of synthesis nearly to the adult levels. The synthesis of choline and ethanolamine phosphoglycerides is not affected in the liver microsomes of ageing rats. The molar distribution of fatty acids in brain microsomal diacyl glycerols of ageing rats is noticeably different from that of adult animals. The content of monoenoic and dienoic species is increased, whereas that of the tetraenoic species is decreased. Base exchange reaction for choline and ethanolamine incorporation into respective phospholipids is not affected in the brain microsomes of the aged rats.     

Effects of dietary protein and cholesterol on phosphatidylcholine and phosphatidylethanolamine molecular species in mouse liver

The present study examined the effects of two atherogenic factors, animal protein and cholesterol, on the distribution of fatty acids and the molecular species of major liver phospholipids in mice. Weanling mice were fed a semisynthetic diet supplemented with either casein or soy protein (20%, w/w) in the presence or absence of 0.5% cholesterol for 4 wk. Results from mouse liver showed that animal protein and, more so, dietary cholesterol modified the fatty acid profiles of the phospholipids. Animal protein had no significant effect on the concentration of lipids, but it altered the relative distribution and fatty acid profiles of the phospholipids, phosphatidylcholine and phosphatidylethanolamine. Dietary cholesterol, on the other hand, significantly increased the concentration of liver lipids, but it did not alter the relative distribution of phosphatidylcholine and phosphatidylethanolamine. In cholesterol-fed mice, the proportions of molecular species containing 18∶2n−6 were increased, whereas those containing 20∶4n−6 were decreased, indicating that dietary cholesterol suppressed linoleic acid metabolism. Since cholesterol feeding selectively decreased the ratio of 18∶0/20∶4n−6 in phosphatidylcholine, whereas it increased the 18∶0/18∶2n−6 ratio in phosphatidylethanolamine, this finding suggests that dietary cholesterol may affect the incorporation of fatty acids but not the rate of synthesis of phosphatidylcholine and phosphatidylethanolamine.     

小鼠肺泡巨噬细胞的分离及鉴定

目的分离小鼠肺泡巨噬细胞(alveolar macrophage,AM),并鉴定其纯度和吞噬活性。方法采用肺泡灌洗法分离小鼠AM,分别通过瑞氏-姬姆萨染色法和Diff-Quik染色法鉴定AM的纯度;将FITC标记的大肠埃希菌与AM按不同比例混合(10∶1、15∶1、20∶1)后孵育不同时间(20、25、30 min),荧光倒置显微镜下观察AM的吞噬活性。结果两种染色方法染色时,阳性细胞百分率均在94%以上,Diff-Quik染色法细胞阳性率略高于瑞氏-姬姆萨染色法,染色时间较瑞氏-姬姆萨染色法短,且背景干净无杂质。小鼠AM的吞噬率和吞噬指数随着FITC标记的细菌与AM比例的增加而增大,且随孵育时间的延长呈先升高再降低的趋势。当细菌与细胞比例为20∶1,孵育时间为25 min时,吞噬率和吞噬指数达最大,分别为(63.67±4.04)%和1.41±0.07。结论贴壁培养法可获得较高纯度的小鼠AM,Diff-Quik染色法鉴定巨噬细胞纯度优于瑞氏-姬姆萨染色法,其可代替瑞氏-姬姆萨染色法,成为一种快速、高效的鉴定巨噬细胞纯度的方法。     

Cytokine release from alveolar macrophages exposed to ambient particulate matter: Heterogeneity in relation to size,city and season

Background  

Several studies have demonstrated an association between exposure to ambient particulate matter (PM) and respiratory and cardiovascular diseases. Inflammation seems to play an important role in the observed health effects. However, the predominant particle component(s) that drives the inflammation is still not fully clarified. In this study representative coarse (2.5–10 μm) and fine (0.1–2.5 μm) particulate samples from a western, an eastern, a northern and a southern European city (Amsterdam, Lodz, Oslo and Rome) were collected during three seasons (spring, summer and winter). All fractions were investigated with respect to cytokine-inducing potential in primary macrophages isolated from rat lung. The results were related to the physical and chemical parameters of the samples in order to disclose possible connections between inflammatory potential and specific characteristics of the particles.     

Comparison of fatty acid and triacylglycerol metabolism of macrophages and smooth muscle cells

The response of macrophages and smooth muscle cells to culture in free fatty acid has been compared. Because oleate and linoleate promoted triacylglycerol enrichment of smooth muscle cells, whereas palmitate had little effect, oleate was used for these studies. The kinetics of the accumulation of triacylglycerol produced by oleate was comparable between smooth muscle cells and macrophages. When grown in increasing concentrations of oleic acid at various fatty acid to albumin molar ratios, the extent of triacylglycerol accumulation in both cell types was dependent on the concentration of oleate, the concentration of albumin, and the oleate to albumin molar ratio. However, macrophages contained 2.6-fold more triacylglycerol than smooth muscle cells in the presence of oleate at 0.36 mM or greater and at levels of albumin higher than 0.15 mM. The cellular triacylglycerol content of macrophages was linearly related to the oleate to albumin molar ratio at both a constant albumin concentration and a constant oleate concentration, whereas the accumulation of triacylglycerol in smooth muscle cells showed a curvilinear relationship. When cells were preloaded with triacylglycerols, smooth muscle cells showed a greater loss of lipid when exposed to albumin than macrophages did. Over a two-hr time period, macrophages incorporated twice as much labeled fatty acid as smooth muscle cells. Thus, while smooth muscle cells and macrophages showed similar responses to exogenous fatty acid and albumin, there were also significant quantitative distinctions.     

Formation of diacyl and alkylacyl glycerophosphocholine in rabbit alveolar macrophages

The incorporation of various labeled precursors into alkenylacyl, alkylacyl and diacyl phospholipids in rabbit alveolar macrophages was studied. The incorporation rates of the individual precursors were shown to be quite different among the three subclasses of phospholipids. [³H] Glycerol, [¹⁴C]16∶0, [¹⁴C]18∶1, [¹⁴C]18∶2 and [³²P]-orthophosphate were preferentially incorporated into choline glycerophospholipids (CGP), especially into diacyl glycerophosphocholine (GPC), indicating that the de novo synthesis of diacyl GPC is extremely high. Considerable portions of the radioactiveties of [¹⁴C]16∶0, [¹⁴C]18∶1, [¹⁴C]18∶2 and [³²P] orthophosphate were also found in alkylacyl GPC, the incorporation being higher than or comparable to that in the case of diacyl glycerophosphoethanolamine (GPE). We then examined the activities of cholinephosphotransferase and ethanolaminephosphotransferase, and found that the activity of cholinephosphotransferase was remarkably high in macrophage microsomes compared with that in microsomes from several other tissues. This suggests that diradylglycerols were preferentially utilized by cholinephosphotransferase, which is consistent with the results obtained for intact cells. We confirmed that a considerably higher amount of diacyl GPC as well as alkylacyl GPC was formed through this enzyme reaction with macrophage microsomes than with brain microsomes. The high formation of alkylacyl GPC could be responsible, at least in part, for the accumulation of this unique ether phospholipid, a stored precursor form of plateletactivating factor in macrophages. Fatty chains are designated in terms of number of carbon atoms: number of double bonds, e.g., 18:1 for oleic acid.     

Mass determination of the fatty acids released from tannin-stimulated rabbit alveolar macrophages

Previous studies with macrophages that had been prelabeled with [¹⁴C]arachidonic acid (20∶4) have shown that condensed tannin is a potent agonist for the release of arachidonic acid. However, it has not been demonstrated that the percentage release of [¹⁴C]20∶4 accurately reflects the metabolic activity of the endogenous 20∶4 pool. In order to measure the 20∶4 mass release relative to the total cellular 20∶4 pool, the free fatty acids of freshly isolated alveolar macrophages were derivatized with a fluorescent reagent, and then separated and quantified by high-performance liquid chromatography. The amounts of esterified fatty acids were measured by gas chromatography of the methyl esters. Free fatty acid levels were compared to those of the total esterified plus unesterified fatty acids to determine the actual percentage released of each fatty acid. Tannin-stimulated release of 20∶4 mass reflected that previously reported for the release of [¹⁴C]20∶4 label but at a slower rate and at a much lower percentage indicating that [¹⁴C]20∶4 had been incorporated into part of a more reactive pool. The specificity of the fatty acid release induced by tannin and β-1,3-glucan, a known agonist for 20∶4 release, was also examined. Both agonists promoted an increase in the levels of free 20∶4 and of other fatty acids. A comparison of the absolute increases of each of the fatty acids indicated that tannin caused a preferential increase in the mass of free 20∶4, whereas β-1,3-glucan evoked a selective increase in the mass of 16∶0. Deceased.     

Hepatic phospholipid molecular species in the guinea pig adaptations to pregnancy

Incorporation of polyunsaturated fatty acids (PUFA), particularly 22∶6n−3, into fetal brain at specific gestational ages is critical for development of normal brain function. We have studied adaptations to maternal liver phospholipid molecular species compositions that may be related to the supply of PUFA to fetal brain. The increment of 22∶6n−3 in brain phosphatidylethanolamine (PE) was maximal at day 25 to day 35 of gestation, consistent with early prenatal development of guinea pig brain. At the same gestational ages, there was a transient increase in maternal liver concentration of 16∶0/22∶6 phosphatidylcholine (PC), which preceded the progressive increase in total PC concentration toward term (day 68). This effect was specific for thesn-1 16∶0 species, as, there was no significant increase in 18∶0/22∶6 PC concentration. These results are consistent with a specific role for 16∶0/22∶6 PC in the directed supply of 22∶6n−3 from maternal liver to the fetus. Concentrations of all PE species in maternal liver decreased at day 25 and day 35 of gestation. The gradual accumulation of 22∶6n−3 in fetal liver throughout gestation did not correlate with the pattern of acquisition of 22∶6n−3 into fetal brain PE. Maternal plasma PC and cholesterol concentrations decreased dramatically by day 25 of gestation, and remained low until term. This hypolipidemia of pregnancy in the guinea pig may be due to increased lipase-mediated turnover of plasma lipoproteins and contrasts strongly with the well-characterized hyperlipidemia in human and rat gestation.     

ROS-mediated TNF-alpha and MIP-2 gene expression in alveolar macrophages exposed to pine dust

BACKGROUND: Respiratory symptoms, impaired lung function, and asthma have been reported in workers exposed to wood dust in a number of epidemiological studies. The underlying pathomechanisms, however, are not well understood. Here, we studied the effects of dust from pine (PD) and heat-treated pine (HPD) on the release of reactive oxygen species (ROS) and inflammatory mediators in rat alveolar macrophages. METHODS: Tumour necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein-2 (MIP-2) protein release, TNF-alpha and MIP-2 mRNA expression, and generation of ROS were studied as end points after treatment of rat alveolar macrophages with PD or HPD. In a separate series of experiments, the antioxidants glutathione and N-acetyl-L-cysteine were included in combination with wood dust. To determine the endogenous oxidative and antioxidant capacity of wood dusts, electron spin resonance (ESR) spectroscopy was used. RESULTS: After 4 h incubation, both PD and HPD elicited a significantly (p < 0.05) increased mRNA expression of TNF-alpha and MIP-2 as well as a concentration-dependent release of TNF-alpha and MIP-2 protein. Interestingly, PD induced a significantly higher TNF-alpha and MIP-2 production than HPD. Moreover, a significantly increased ROS production was observed in alveolar macrophages exposed to both PD and HPD. In the presence of the antioxidants glutathione and N-acetyl-L-cysteine, the PD- and HPD-induced release of ROS, TNF-alpha, and MIP-2 was significantly reduced. Finally, electron spin resonance analyses demonstrated a higher endogenous antioxidant capacity of HPD compared to PD. Endotoxin was not present in either dust sample. CONCLUSION: These results indicate that pine dust is able to induce expression of TNF-alpha and MIP-2 in rat alveolar macrophages by a mechanism that is, at least in part, mediated by ROS.     

Dietary fats containing concentrates ofcis ortrans octadecenoates and the patterns of polyunsaturated fatty acids of liver phosphatidylcholine and phosphatidylethanolamine

The effects of the mixedcis- 18∶1 isomers and mixedtrans-18∶1 isomers present in partially hydrogenated soybean oil (PHSO) upon the patterns of polyunsaturated fatty acids (PUFA) in liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were studied in rats fed concentrates ofcis- 18∶1 ortrans- 18∶1 isomers isolated as triacylglycerides from PHSO. Thecis- 18∶1 andtrans- 18∶1 concentrates were fed at levels equal to those present in PHSO fed at 17.9% of the diet. All diets contained the required amounts of both linoleic and linolenic acids. Thetrans- 18∶1 concentrate was found to suppress the levels of 20∶4ω6 and 20∶3ω9, and to increase the levels of 18∶2ω6 and 20∶5ω3 in PC and PE. Thecis- 18∶1 concentrate suppressed 20∶4ω6 in PC, 20∶5ω3 in PC and PE, and 18∶2ω6 was more effective than thetrans concentrate in suppressing 22∶6ω3. Thetrans- 18∶1 concentrate was more effective in suppressing 20∶4ω6. Thetrans-18∶ isomers appear to modify PUFA metabolism by inhibition of PUFA synthesis, whereas thecis- 18∶1isomers appear to compete with 2-position fatty acyl transfer and to inhibit ω3 PUFA acylation.     

Regulation of the biosynthesis of platelet-activating factor in alveolar macrophages

Activities of enzymes which metabolize lysoplatelet-activating factor (lysoPAF) and platelet-activating factor (PAF) were studied in rabbit alveolar macrophage lysates. Substantial acetyltransferase activity was noted in the presence of 100 μM acetyl-coenzyme A (CoA), and this activity was increased in A23187-stimulated cell lysate. On the other hand, in the absence of exogenous acetyl-CoA, lysoPAF was mainly acylated through a transacylation pathway rather than by acetyltransferase in both control and A23187-stimulated cell lysates. We confirmed that the intracellular concentration of acetyl-CoA is relatively low. The observations suggest that the transacylation system may play an equally important role in the regulation of the availability of lysoPAF in intact cells. Intracellular lysoPAF was also maintained at relatively low levels. Interestingly, large amounts of PAF were produced even in unstimulated cells upon addition of an excess of exogenous lysoPAF, suggesting that generation of an adequate amount of lysoPAF within cells may be sufficient to trigger PAF synthesis in this type of cells. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

ROS-mediated TNF-α and MIP-2 gene expression in alveolar macrophages exposed to pine dust

Background  

Respiratory symptoms, impaired lung function, and asthma have been reported in workers exposed to wood dust in a number of epidemiological studies. The underlying pathomechanisms, however, are not well understood. Here, we studied the effects of dust from pine (PD) and heat-treated pine (HPD) on the release of reactive oxygen species (ROS) and inflammatory mediators in rat alveolar macrophages.