Neutrophil Phospholipids Research Articles

Time-dependent utilization of platelet arachidonic acid by the neutrophil in formation of 5-lipoxygenase products in platelet-neutrophil co-incubations

The biosynthesis of leukotrienes is known to occur through a series of complex processes which in part, can be influenced by cell-cell interactions. Several studies have suggested that arachidonic acid availability is a major limiting step for leukotriene biosynthesis and that its transfer between cells can represent a significant source of this precursor. Accordingly, effect of time and source of arachidonic acid on transcellular leukotriene synthesis was studied in mixed platelet/neutrophil populations challenged with the calcium ionophore A23187. A time-dependent contribution of platelet-derived as well as neutrophil-derived arachidonate was found in the selective formation of neutrophil 5-lipoxygenase metabolites. Utilization of platelet or neutrophil arachidonate was followed by incorporation of radiolabeled arachidonic acid into platelet or neutrophil phospholipids prior to stimulation. Specific activity of liberated arachidonic acid along with numerous 5-lipoxygenase products (including LTB 4, 20-hydroxyl-LTB 4, 5-HETE and LTC 4) was determined in order to follow mass and radiolabel. A large amount of platelet-derived arachidonic acid was released in the first 1.5 min, whereas 10 min platelet-derived arachidonate was much lower in amount but significantly higher in specific activity, suggesting different precursor pools. The platelet-derived arachidonate was heavily utilized by the neutrophils at the early time points for formation of 5-HETE and Δ 6- trans-LTB 4 isomers, but appeared to contribute only marginally to the constitutive metabolism of neutrophil arachidonate into LTB 4. Results from these experiments suggest different pools of 5-lipoxygenase in the neutrophil and indicate a time and source dependent modulation of arachidonate metabolism in mixed cell interactions.

Linoleate inhibits EPA incorporation from dietary fish-oil supplements in human subjects

Thirty healthy male subjects were randomly allocated into one of two treatment groups: group H subjects were maintained on a high-linoleic acid (LA) diet with the saturated fatty acids kept low and those in group L were kept on a low-LA, low saturate diet. After a 3-wk run-in period, subjects consumed a fish-oil supplement containing 1.6 g eicosapentaenoic acid (EPA)/d and 0.32 g docosahexaenoic acid as triglycerides for a further 4 wk. The diets alone resulted in a significant change in neutrophil phospholipid LA (H = 12.7 +/- 2.9%, L = 9.0 +/- 0.9%; P less than 0.05). Dietary supplementation with fish oil resulted in a significant increase in EPA in neutrophil phospholipids that was greater in group L (mean 2.0 +/- 0.4%) than group H (mean 1.5 +/- 0.3%; P less than 0.005). Reductions in arachidonic acid concentrations in neutrophil membranes were not different, nor could differences in leukotriene B4 production be detected. Our study indicates that the ingestion of n-6 fatty acids within the diet is an important determinant of EPA incorporation into neutrophil membranes.

Supplementation with evening primrose oil in atopic dermatitis: Effect on fatty acids in neutrophils and epidermis

We investigated the effect of oral supplementation with evening primrose oil, containing 72% linoleic acid (18:2n-6) and 10% gamma-linolenic acid (18:3n-6), on the epidermal and neutrophil phospholipid fatty acid composition in 15 patients with atopic dermatitis (AD). Three different dose levels, 4, 8 and 12 capsules per day containing 0.5 g oil, were given to three groups of patients. The only n-6 fatty acid showing a significant (p less than 0.05) dose-related increase was dihomo-gamma-linolenic acid (20:3n-6) in neutrophil phospholipids. The highest dose increased dihomo-gamma-linolenic acid by 45% in neutrophil phospholipids, by 46% in lesion-free epidermal phosphatidylcholine, and by 15% in lesion-free epidermal phosphatidylethanolamine. In both lesional and lesion-free epidermis, supplementation resulted in a rise in the ratio between n-6 and monounsaturated fatty acids, reaching significance (p less than 0.05) in lesional epidermis. This study shows that moderate and favorable fatty acid changes can be obtained in the epidermis of AD patients, when given 6 g per day of oil rich in n-6 fatty acids. The abnormal lipid and fatty acid pattern of the atopic epidermis may be involved in the pathogenesis of the disease, and should therefore be the target for future therapeutic approaches with fatty acid supplements.

Essential fatty acids and serine as plasmalogen precursors in relation to competing metabolic pathways

Interest in altered ether-lipid metabolism, associated with peroxisomal disorders including adrenoleukodystrophy and Zellweger's syndrome, has highlighted present limitations in our understanding of the biosynthesis and turnover of plasmalogens. These 1-alkenyl ethanolamine phosphoglycerides are major phospholipids in brain, vascular tissue, neutrophils, and most tumors, and they constitute 15-20% of total phospholipids in cultured glioma cell. In glioma, turnover of polyunsaturated acyl chains in the sn-2 position of plasmalogens was examined in relation to selectivity for the (n - 3) and (n - 6) families. Remodeling of acyl chains was more dependent on chain length than on selectivity between families, consistent with plasmalogens enriched in polyunsaturated, but not specifically (n - 3), fatty acids. Extracellular serine was a precursor of serine and ethanolamine phosphoglycerides and was associated with plasmalogens due to decarboxylation and headgroup exchange. Incorporation of extracellular serine ceased within 8 h, even though more than 50% of the label remain in the medium. Analyses of medium and cellular water-soluble components indicated rapid conversion of serine to glycine and other metabolites not used in phospholipid biosynthesis. Thus, nutrient molecules as precursors of plasmalogens are involved in complex competitive interactions. As functions of plasmalogens are clarified, regulation of plasmalogen turnover becomes an increasingly important issue and elucidation of these processes is essential.

Leukotriene B4 promotes phospholipid acylation in human neutrophils.

The present study was undertaken to test the hypothesis that leukotriene B4 (LTB4) may promote extracellular fatty acid incorporation into neutrophil choline glycerophospholipids (PC) to replenish phospholipids after deacylation. Incubation of human neutrophils with LTB4 (1.5 to 150 nM) for 1 to 5 min resulted in increased fatty acid incorporation into phosphatidylinositol (PI), diacyl-sn-glycero-3-phosphocholine (diacyl-GPC) and alkylacyl-GPC. The magnitude of stimulation (percentage of control) of fatty acid incorporation appears to reflect increased activity of the acyltransferase catalyzing acylation of the respective lysophospholipids. LTB4 stimulation of arachidonic acid incorporation into PI was greater than into PC, whereas the stimulation of palmitic acid incorporation into PC was greater than into PI. LTB4 stimulated phosphatidic acid labeling by palmitic acid but not by arachidonic acid. LTB4 and 1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (cPAF) exhibited a similar stimulatory effect on fatty acid incorporation into the PC fraction. Phosphate analysis could not detect changes in the mass of PI or of PC in neutrophils exposed to LTB4 or cPAF. The results suggest that increased fatty acid incorporation into phospholipids in LTB4-activated neutrophils reflects activation of phospholipase A2 and acyltransferases as well as of de novo phospholipid synthesis.

Effects of a fish oil diet on the metabolism of endogenous ( n−6) and ( n−3) fatty acids in rat neutrophils

The present study was carried out to define more precisely how dietary ( n−3) fatty acids mediate leukotriene and phospholipid metabolism in neutrophils. Neutrophils from chow-fed rats did not synthesize detectable levels of LTB 5 or 5-hydroxyeicosapentanenoic acid. The ratio of esterified 20:4(n−6) 20:5(n−3) in the phospholipids of neutrophils from rats fed a corn oil /fish oil diet was 4.6. The ratio of LTB 4/LTB 5 made by these cells was 2.6, thus suggesting that 20:5( n−3) release and/or subsequent metabolism was somewhat more efficient than for 20:4( n−6). When tritiated lyso-platelet activating factor was added to neutrophils from chow-fed rats, it was deactivated primarily by acylation with arachidonic acid. With the fish oil-fed animals both arachidonic acid and 20:5( n−3) were transferred to deactivate lyso-platelet activating factor. Molecular species analysis of the resulting radioactive 1- O-alkyl-2-acyl- sn-glycerol-3-phosphocholine showed that 20:5( n−3) pairs with the same 1- O-alkyl groups and in approximately the same ratio as does arachidonate. Collectively, these studies show that once 20:5( n−3) is incorporated into neutrophil lipids it is metabolized in a similar way as is arachidonate.

Effects of dietary supplementation with eicosapentaenoic acid or gamma-linolenic acid on neutrophil phospholipid fatty acid composition and activation responses

Previous data that alimentation with fish oil rich in eicosapentaenoic acid (EPA; 20:n-3) or vegetable oil rich in gamma-linolenic acid (GLA; 18:3n-6) can reduce symptoms of inflammatory skin disorders lead us to determine the effects of dietary supplements of oils rich in EPA or GLA on guinea pig (GP) neutrophil (PMN) membrane potential (delta gamma), secretion, and superoxide (O2-) responses. Weanling GPs were initially fed diets supplemented with olive oil (less than 0.1% EPA; less than 0.1% GLA) for 2 weeks, followed by a crossover by two sets of animals to diets supplemented with fish oil (19% EPA) or borage oil (25% GLA). At 4-week intervals, 12% sterile casein-elicited peritoneal neutrophils (PMN) were assessed for membrane polyunsaturated fatty acid (PUFA) profiles and FMLP-, LTB4-, and PMA-stimulated delta gamma changes, changes in flow cytometrically measured forward scatter (FWD-SC) (shape change), 90 degrees scatter (90 degrees -SC) in cytochalasin B-pretreated-PMN (secretion response), and superoxide responses, GP incorporated EPA and GLA (as the elongation product, dihomo-GLA or DGLA) into their PMN phospholipids by 4 weeks. The peritoneal PMN of all groups demonstrated broad resting FWD-SC and poor activation-related FWD-SC increases, suggesting in vivo activation. While secretion was comparable in the three groups in response to FMLP, there was a trend toward inhibition of LTB4-stimulated 90 degrees -SC loss in both fish and borage oil groups. This was significant only with borage oil (21.7 +/- 2.1 vs 15.3 +/- 1.2% loss of baseline 90 degrees -SC, olive vs borage: P = 0.03). PMN from borage- and fish oil-fed GPs showed a progressively lower O2- response to FMLP than the olive oil group (73.9 +/- 3.9 and 42.9 +/- 6.8% of olive oil response for borage and fish oils, respectively; P less than 0.005 and P less than 0.01, respectively, at 12 weeks), while PMA-stimulated O2- was inhibited only in the fish oil-fed group and only at 12 weeks (62.0 +/- 2.7% of control; P less than 0.025). We conclude that dietary supplementation with oils rich in PUFAs can modify PMN activation responses.

Catabolism of leukotriene B5 in humans

Human neutrophils, enriched by dietary supplementation with eicosapentaenoic acid, form leukotriene (LT)B5 in addition to LTB4 upon stimulation. LTB5 is one order of magnitude less biologically active than the potent chemokinetic and chemoattractant LTB4. Catabolites of LTB5 have not yet been characterized in vitro and ex vivo. It is unknown whether catabolism of LTB5 interferes with catabolism of LTB4. This report describes catabolism of LTB5 to 20-OH-LTB5, which in turn is catabolized to 20-COOH-LTB5. The structures of the two catabolites were established by UV-absorbance, behavior on reverse-phase high-performance liquid chromatography, enzymatic analysis of human neutrophils, and gas chromatography-mass spectrometry. In vitro, formation of LTB4 was delayed and formation of its catabolites was depressed by exogenous eicosapentaenoic acid. By supplementing the diet of six volunteers with 5 g eicosapentaenoic acid/day for 7 days, eicosapentaenoic acid quadrupled in neutrophil phospholipid fatty acids. Consequently, LTB5, 20-OH-LTB5, and 20-COOH-LTB5 were detected ex vivo. In contrast to the findings in vitro, however, levels of LTB4, 20-OH-LTB4, and 20-COOH-LTB4 were unaltered by the dietary intervention. Thus, in vitro, but not ex vivo, addition of eicosapentaenoic acid, and subsequent formation of LTB5, impeded catabolism of proinflammatory LTB4.

Effects of fish oil on serum lipids in men during a controlled feeding trial

Effects of fish-oil (FO) feeding on serum lipids were investigated in a 42-d controlled diet study. Fifteen healthy male college students were assigned to one of three groups: control (0 g FO); 5 g FO, supplying 2 g n - 3 (omega-3) fatty acids (FAs); or 20 g FO, supplying 8 g n - 3 FAs. In an initial 7-d period subjects consumed a basal diet with no FO. Then FO replaced an equivalent amount of margarine for 5 wk. FO feeding significantly (p less than 0.05) decreased the serum n - 6 FAs, linoleic acid, eicosatrienoic acid, and arachidonic acid. A significant increase in the n - 3 FAs, eicosapentaenoic acid and docosahexaenoic acid, was noted in serum, platelet, and neutrophil phospholipids. The 20-g-FO group showed a 30% decrease (p less than 0.01) in triglycerides after 2 wk FO with no further decrease observed. Thus, 20 g FO produced changes in both FA patterns and triglyceride concentrations whereas 5 g FO produced changes in FA patterns only. Neither FO amount resulted in significant changes in total or HDL cholesterol, apolipoprotein A-I, or apolipoprotein B-100.

Lipoxin A4 and lipoxin B4 stimulate the release but not the oxygenation of arachidonic acid in human neutrophils: Dissociation between lipid remodeling and adhesion

The profiles of actions of lipoxin A4 (LXA4) and lipoxin B4 (LXB4), two lipoxygenase-derived eicosanoids, were examined with human neutrophils. At nanomolar concentrations, LXA4 and LXB4 each stimulated the release of [1-14C]arachidonic acid from esterified sources in neutrophils. Lipoxin-induced release of [1-14C]arachidonic acid was both dose- and time-dependent and was comparable to that induced by the chemotactic peptide f-met-leu-phe. Time-course studies revealed that lipoxin A4 and lipoxin B4 each induced a biphasic release of [1-14C]arachidonic acid, which was evident within seconds (5-15 sec) in its initial phase and minutes (greater than 30 sec) in the second phase. In contrast, the all-trans isomers of LXA4 and LXB4 did not provoke [1-14C]AA release. Lipoxin-induced release of arachidonic acid was inhibited by prior treatment of the cells with pertussis toxin but not by its beta-oligomers, suggesting the involvement of guaninine nucleotide-binding regulatory proteins in this event. Dual radiolabeling of neutrophil phospholipid classes with [1-14C]arachidonic acid and [3H]palmitic acid showed that phosphatidylcholine was a major source of lipoxin-induced release of [1-14C]arachidonic acid. They also demonstrated that lipoxins rapidly stimulate both formation of phosphatidic acid as well as phospholipid remodeling. Although both LXA4 and LXB4 (10(-8)-10(-6) M) stimulated the release of [1-14C]arachidonic acid, neither compound evoked its oxygenation by either the 5- or 15-lipoxygenase pathways (including the formation of LTB4, 20-COOH-LTB4, 5-HETE, or 15-HETE). LXA4 and LXB4 (10(-7) M) each stimulated the elevation of cytosolic Ca2+ as monitored with Fura 2-loaded cells, albeit to a lesser extent than equimolar concentrations of FMLP. Neither lipoxin altered the binding of [3H]LTB4 to its receptor on neutrophils. In addition, they did not stimulate aggregation or induce adhesion of neutrophils to human endothelial cells. Results indicate that both LXA4 and LXB4 stimulate the rapid remodeling of neutrophil phospholipids to release arachidonic acid without provoking either aggregation or the formation of lipoxygenase-derived products within a similar temporal and dose range. Together they indicate that LXA4 and LXB4 display selective actions with human neutrophils and suggest that these eicosanoids possess unique profiles of action which may regulate neutrophil function during inflammation.

Studies on the incorporation and transacylation of various fatty acids in choline and ethanolamine-containing phosphoacyglycerol subclasses in human neutrophils

The incorporation of eight 14C-labeled fatty acids into human neutrophil phospholipids was investigated and the results were expressed as percent of the total phospholipid associated 14C-labeled substrate incorporated after an initial 15 min labeling and a subsequent 2 h reincubation in fatty acid-free buffer. In all cases, the PC fraction accounted for more than 40% of the total phospholipid radioactivity. The inositol-containing phosphoacylglycerols were also labeled well by all the fatty acids except 22:6(n - 3) and 16:0; however, a greater percentage of [14C]22:6(n - 3) was found in PE than that of any other labeled fatty acid substrate. In all cases, most of the radioactivity in PC after 15 min was in the diacyl subclass. After 2 h, there was a shift of [14C]20:4(n - 6), [14C]20:5(n - 3), [14C]22:6(n - 3) and [14C]18:4(n - 4) into the ether-linked subclass. No such shift was observed for [14C]16:0 or [14C]18:2(n - 6) and, although there was an increase in the percent radioactive 20:3(n - 6) and 20:3(n - 9) in ether-linked PC after 2 h, the total radioactivity in this fraction remained low by comparison. A similar shift in label occurred in the plasmalogenic-linked PE subspecies in cells labeled with [14C]20:4(n - 6), [14C]20:5(n - 3) and [14C]22:6(n - 3).

Quantitative analysis of phospholipids and demonstration of plasmalogen in human neutrophil subcellular fractions by high-performance liquid chromatography.

The subcellular distribution of phospholipids in unstimulated neutrophils was investigated by high-performance liquid chromatography of lipid extracts of granule and plasma membranes obtained from Percoll density gradients. The mobile phase contained acetonitrile:methanol:85% phosphoric acid (131:3:0.8 v/v/v). Post-nuclear supernatants contained 2.3 micrograms lipid phosphorus/mg protein. Phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine were demonstrated in azurophil granules, specific granules and in a combined fraction of secretory granules and plasma membranes. Separate estimates of each phospholipid class by peak areas obtained by high-performance liquid chromatography showed that secretory granules and plasma membranes contained most of the phosphatidylinositol and phosphatidylcholine (p less than 0.005 vs peaks areas obtained in azurophil and specific granules), whereas a major part of the phosphatidylethanolamine was located in the specific granules (p less than 0.005 vs peak areas obtained in azurophil granules, and plasma membrane and secretory granules). High-performance liquid chromatography proved to be a useful principle for the demonstration of plasmalogen because the acidic solvent caused hydrolysis of phosphatidalethanolamine, which was recovered as lysophosphatidylethanolamine. Additionally, sphingomyelin was demonstrated in all subfractions by thin-layer chromatography.

Reacylation of platelet activating factor with eicosapentaenoic acid in fish-oil-enriched monkey neutrophils

Platelet activating factor (PAF) is rapidly metabolized via a deacetylation: reacylation pathway which shows striking specificity for arachidonate at the sn-2 position of the 1- O-alkyl-2-acyl-GPC thus formed. We have now examined the effects of a diet enriched in fish oils on the metabolism of PAF and specificity for arachidonate in the reacylation reaction. [ 3H]PAF was incubated for various lengths of time with neutrophils from monkeys fed a control diet or one enriched in fish oils. The [ 3H]PAF added to the cell suspension was rapidly converted to 1- O-alkyl-2-acyl-GPC. Reverse-phase HPLC analysis of the acyl chains added at the sn-2 position revealed that arachidonate was the major fatty acid incorporated into the 1- O-alkyl-2-acyl-GPC formed by neutrophils from monkeys on the control diet. In contrast, both 1- O-alkyl-2-arachidonoyl-GPC and 1- O-alkyl-2-eicosapentaenoyl-GPC were formed by the fish-oil-enriched neutrophils. We also report on the fatty acid composition of neutrophil phospholipids during such a diet.

The effect of calcium ionophore A23187 on the metabolism of arachidonic and eicosapentaenoic acids in neutrophils from a marine teleost fish rich in (n-3) polyunsaturated fatty acids

1. 1. [1- 14C]AA and [1- 14C]EPA were incorporated equally into plaice neutrophil phospholipids. 2. 2. Incubation with A23187 resulted in the loss of label from total phospholipids and increased label released from the neutrophils. 3. 3. Labelled LTB 4 and LTB 5 production was increased 2.5- and 3-fold, respectively, by A23187 treatment. 4. 4. However, the incorporated AA was generally more metabolically active than the incorporated EPA with approx. 3–4 times as much LTB 4 produced than LTB 5 by cells in the presence or absence of A23187, respectively. 5. 5. The results obtained in this (n-3) PUFA-rich species were discussed in comparison with current knowledge of AA and EPA metabolism in mammalian neutrophils.

Incorporation of [1- 14C]arachidonic and [1- 14C]eicosapentaenoic acids into the phospholipids of peripheral blood neutrophils from the plaice, Pleuronectes platessa L.

The incorporation of [1- 14C]arachidonic acid and [1- 14C]eicosapentaenoic acids into phospholipids was studied in peripheral blood neutrophils from plaice, a marine fish whose lipids are rich in ( n — 3) polyunsaturated fatty acids. The incorporation of both labelled fatty acids into phospholipids was approximately equal when presented individually, and at equal concentration, to the cells. Arachidonic acid was relatively preferred when both acids were present in the incubations. When incorporation was expressed per unit mass of phospholipid class, the order of incorporation was PI > PC > PE > PS > sphingomyelin for both fatty acids. However, the specificity for incorporation into PI was significantly greater with arachidonic acid. Eicosapentaenoic acid was incorporated into PC to a greater extent than arachidonic acid. The results are discussed in relation to the possible roles of arachidonic acid and eicosapentaenoic acid in polyunsaturated fatty acid metabolism in plaice neutrophils.

Leukotriene B formation by neutrophils from essential fatty acid-deficient rats.

Analysis of neutrophil phospholipids from rats fed an essential fatty acid-deficient diet revealed a 33% reduction in arachidonate and a 90% reduction in linoleate compared to neutrophil phospholipids of rats fed a normal diet. The neutrophil phospholipids from rats fed the essential fatty acid-deficient diet also contained significant amounts of 5,8,11-eicosatrienoate, a fatty acid not found in the neutrophils of rats fed a normal diet. Analysis of the production of leukotrienes of the B series by ionophore-stimulated neutrophils from rats fed an essential fatty acid-deficient diet revealed a 87% reduction in leukotriene B4 compared to neutrophils from rats fed a normal diet even though the arachidonate content was reduced by only 34%. Essential fatty acid-deficient neutrophils converted endogenous 5,8,11-eicosatrienoic acid to leukotriene A3 and its nonenzymatic degradation products, but little or no leukotriene B3 was formed. Neutrophils from rats fed a normal diet incubated with ionophore and exogenous 5,8,11-eicosatrienoate also produced leukotriene A3 and its nonenzymatic degradation products but little or no leukotriene B3. Exogenous 5,8,11-eicosatrienoate incubated with ionophore-stimulated normal neutrophils caused a dose-dependent inhibition of leukotriene A hydrolase resulting in diminished production of leukotriene B4 from endogenous arachidonate. Assays of leukotriene A hydrolase in the 10,000 X g supernatant fraction of a homogenate of RBL-1 cells revealed that a lipoxygenase metabolite of 5,8,11-eicosatrienoate rather than 5,8,11-eicosatrienoate itself is the inhibitor of leukotriene A hydrolase. Thus the finding that leukotriene B4 production by neutrophils from essential fatty acid-deficient rats is diminished out of proportion to the decrease in arachidonate content appears to be due to inhibition of leukotriene A hydrolase by a lipoxygenase metabolite.

Uptake, release and metabolism of docosahexaenoic acid (DHA, C22:6ω3) in human platelets and neutrophils

Exogenous DHA is converted by human platelets to 14- and 11- HDHE and by human neutrophils mainly to 7- HDHE . Human platelets prelabeled with 14C-DHA, 14C-EPA and 14C-AA and stimulated with thrombin release and metabolize DHA only in trace amounts as compared to EPA and AA. 14C-DHA is incorporated into the 2-position of platelet phospholipids and occurs predominantly in phosphatidylethanolamine. DHA and EPA were also incorporated by dietary means into phospholipids of platelets and neutrophils. In resting platelets free DHA as well as free AA and EPA are not detectable. In platelets stimulated ex vivo with thrombin DHA is not significantly released which is in contrast to EPA and AA. After stimulation, 14- HDHE is found only in trace amounts as compared to 12-HETE and 12- HEPE . In DHA enriched neutrophils formation of HDHEs cannot be demonstrated after stimulation with ionophore A 23187. We conclude that even after dietary enrichment of DHA in phospholipids of platelets and neutrophils the level of free DHA and/or formation of HDHEs might be too low to substantially affect arachidonic acid metabolism and related functions of these cells.

Inhibition of leukocyte chemotaxis by Glu-Glu-Glu-Glu-Tyr-Pro-Met-Glu and Leu-Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gln-Gly

Chemotaxis of rabbit peritoneal leucocytes stimulated by fMet-Leu-Phe, a synthetic chemoattractant, was inhibited by Glu-Glu-Glu-Glu-Tyr-Pro-Met-Glu (MT peptide) and Leu-Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Glu-Gly (Src peptide). Both peptides did not inhibit the binding of [ 3H] formyl-NLe-Leu-Phe, a chemoattractant, to neutrophils, suggesting that the peptides inhibit the events distal to the chemotactic receptors. These peptides blocked the release of arachidonic acid from phospholipids in neutrophils stimulated with chemoattractants, whereas they had no effect on phospholipase A 2 activity itself. The peptides markedly reduced the phosphorylation of lipomodulin, a phospholipase inhibitory protein, in either intact cells or isolated plasma membranes. Lipomodulin immunoprecipitated by monoclonal anti-lipomodulin antibody had phosphorylserine and phosphoryltyrosine as analyzed upon electrophoresis. The MT peptide which does not contain threonine or serine was phosphorylated by isolated plasma membranes. These results, taken together, suggest that a tyrosine phosphorylating kinase is involved in biochemical events of chemotactic receptors, and that lipomodulin is a substrate for this kinase.

Arachidonic acid metabolism in polymorphonuclear leukocytes from patients with chronic granulomatous disease.

The effect of the calcium ionophore A23187 on the release and metabolism of [3H]arachidonic acid was examined in normal polymorphonuclear leukocytes and those obtained from patients with chronic granulomatous disease. The ionophore A23187 which stimulates oxidative metabolism in normal polymorphonuclear leukocytes was ineffective in increasing oxidative metabolism (chemiluminescence) in polymorphonuclear leukocytes from patients with chronic granulomatous disease. However, the ionophore A23187 stimulated the release of [3H]arachidonic acid from chronic granulomatous disease neutrophil phospholipids and stimulated its metabolism into hydroxyeicosatetraenoic acids and leukotrienes.

Mechanism of arachidonic acid release in human polymorphonuclear leukocytes

Previous studies have demonstrated that [ 3H]arachidonic acid is released from prelabeled human neutrophil phospholipids when the cells are stimulated by calcium ionophore A23187 or by opsonized zymosan. Neither lysophospholipid generated by phospholipase A 2 activity, diacylglycerol nor monoacylglycerol produced via phospholipase C/diacylglycerol lipase action have been identified following neutrophil challenge. The inability to detect any intermediates during the release of arachidonate is due to either rapid reacylation of lysophospholipid or conversion of diacylglycerol (monoacylglycerol) to cellular acylglycerols. The addition of exogenous [ 14C]fatty acid at the time of challenge was employed to determine the involvement of either phospholipase A 2 or phospholipase C activities. Neutrophil stimulation with calcium ionophore A23187 resulted in an incorporation of exogenous [ 14C]arachidonate into phosphatidylinositol and phosphatidylcholine, those phospholipids which specifically release arachidonate. When the saturated fatty acid, [ 14C]stearate, replaced [ 14C]arachidonate, very little [ 14C]fatty acid was incorporated into any of the phospholipid species. Lipid phosphorus measurements revealed no significant mass change in any phospholipid class following ionophore challenge. Production of [ 14C]phosphatidic acid was not detected, as would be expected if diacylglycerol kinase and de novo phospholipid metabolism were significantly involved.