Neutrophil Arachidonic Acid Research Articles

Neutrophil generation of inflammatory precursors is not modulated by docosahexaenoic acid

It is believed that correction of membrane fatty acid deficiency in cystic fibrosis (CF) downregulates the synthesis of proinflammatory mediators. We tested the hypothesis that an increase of the proportion of docosahexaenoic acid (DHA) in the membrane in vitro changes the neutrophil response to Pseudomonas aeruginosa lipopolysaccharide (LPS). Treatment with DHA increased the secretion of interleukin(IL)-1|*alpha*| by CF neutrophils, but the secretion of other cytokines, CD11b expression, and arachidonic acid (AA) release were not affected either in CF or control (CT) neutrophils. Both with and without DHA, only one out of eight CF neutrophils responded to LPS with an increase of released AA, while five out of seven CT cells released more AA (CF vs. CT P < 0.05 by Fisher test). These results indicate that in neutrophils the beneficial effects of DHA on immune response are not directly related to the generation of proinflammatory precursors, and suggest that in CF the lower neutrophil AA generation in response to activation could cause insufficient production of lipid mediators involved in the resolution of lung inflammation.

Cytosolic Phospholipase A2-α Is Necessary for Platelet-activating Factor Biosynthesis, Efficient Neutrophil-mediated Bacterial Killing, and the Innate Immune Response to Pulmonary Infection: cPLA2-α DOES NOT REGULATE NEUTROPHIL NADPH OXIDASE ACTIVITY

The role of a cytosolic phospholipase A(2)-alpha (cPLA(2)-alpha) in neutrophil arachidonic acid release, platelet-activating factor (PAF) biosynthesis, NADPH oxidase activation, and bacterial killing in vitro, and the innate immune response to bacterial infection in vivo was examined. cPLA(2)-alpha activity was blocked with the specific cPLA(2)-alpha inhibitor, Pyrrolidine-1 (human cells), or by cPLA(2) -alpha gene disruption (mice). cPLA(2)-alpha inhibition or gene disruption led to complete suppression of neutrophil arachidonate release and PAF biosynthesis but had no effect on neutrophil NADPH oxidase activation, FcgammaII/III or CD11b surface expression, primary or secondary granule secretion, or phagocytosis of Escherichia coli in vitro. In contrast, cPLA(2)-alpha inhibition or gene disruption diminished neutrophil-mediated E. coli killing in vitro, which was partially rescued by exogenous arachidonic acid or PAF but not leukotriene B(4). Following intratracheal inoculation with live E. coli in vivo, pulmonary PAF biosynthesis, inflammatory cell infiltration, and clearance of E. coli were attenuated in cPLA(2)-alpha(-/-) mice compared with wild type littermates. These studies identify a novel role for cPLA(2)-alpha in the regulation of neutrophil-mediated bacterial killing and the innate immune response to bacterial infection.

Eicosanoids in sickle cell disease: Potential relevance of neutrophil leukotriene B4 to disease pathophysiology

Neutrophil activation with the release of intracellular granule contents has been observed in sickle cell disease (SCD). Because leukotriene B4 (LTB4), a 5-lipoxygenase metabolite of arachidonic acid in neutrophils, is a chemoattractant and enhances neutrophil adhesion to endothelium, we assessed plasma levels of this metabolite in controls (n = 9) and individuals with SCD, SS genotype, both in basal “steady state” (n = 37) and during episodes of vaso-occlusion (n = 10) and acute chest syndrome (n = 5). Thirteen patients with SCD, SC genotype, in steady state were also studied. Although no significant differences were noted between the control (136 ± 32 fmol/mL) and SC genotype (177 ± 83 fmol/mL, P >.15), LTB4 levels were markedly increased in patients with SS genotype in basal steady state (207 ± 64 fmol/mL, P <.003) compared with those in controls. Values were further increased during vaso-occlusion (264 ± 94 fmol/mL) and acute chest syndrome (363 ± 124 fmol/mL). These levels were significantly different from measurements taken during steady state (P <.04 and P <.0001, respectively). No correlation was noted between LTB4 level and total white cell or neutrophil count. Additionally, the significant correlation noted in SCD between increased levels of plasma LTB4 and soluble L-selectin (P <.03) reflects neutrophil activation. We also observed an effect of LTB4 on red cell-endothelial adhesion at concentrations that appear clinically relevant (1-10 pmol/mL) with concomitant up-regulation of mRNA for the endothelial vitronectin receptor. These properties of LTB4 are relevant to disease pathophysiology, providing further evidence of the contribution of the neutrophil to the proinflammatory and proadhesive phenotype in SCD. (J Lab Clin Med 2002;139:80-9)

This month in J Lab Clin Med: Issue highlights for February 2002

This month in J Lab Clin Med: Issue highlights for February 2002

Involvement of cytosolic phospholipase A2 and secretory phospholipase A2 in arachidonic acid release from human neutrophils.

The purpose of this study was to define the role of secretory phospholipase A2 (sPLA2), calcium-independent PLA2, and cytosolic PLA2 (cPLA2) in arachidonic acid (AA) release from fMLP-stimulated human neutrophils. While fMLP induced the release of extracellular sPLA2 activity and AA, 70% of sPLA2 activity remained associated with the cell. Treatment with the cell-impermeable sPLA2 inhibitors DTT or LY311-727, or the anti-sPLA2 Ab 3F10 all inactivated extracellular sPLA2 activity, but had minimal effect on neutrophil AA mass release. In contrast, coincubation of streptolysin-O toxin-permeabilized neutrophils with DTT, LY311-727, or 3F10 all decreased [3H8]AA release from [3H8]AA-labeled, fMLP-stimulated cells. Exposure to fMLP resulted in a decrease in the electrophoretic mobility of cPLA2, a finding consistent with cPLA2 phosphorylation, and stimulated the translocation of cPLA2 from cytosolic to microsomal and nuclear compartments. The role of cPLA2 was further evaluated with the cPLA2 inhibitor methyl arachidonyl fluorophosphonate, which attenuated cPLA2 activity in vitro and decreased fMLP-stimulated AA mass release by intact neutrophils, but had no effect on neutrophil sPLA2 activity. Inhibition of calcium-independent PLA2 with haloenol lactone suicide substrate had no effect on neutrophil cPLA2 activity or AA mass release. These results indicate a role for cPLA2 and an intracellular or cell-associated sPLA2 in the release of AA from fMLP-stimulated human neutrophils.

The role of the endogenous anti-inflammatory compound gravidin in pre-eclampsia

Objectives: The study’s aims were to investigate the levels of gravidin, an endogenous phospholipase A 2 inhibitor, in pregnancy and pre-eclampsia and to establish its effects on neutrophil function. Study Design: Serum samples were collected from 9 nonpregnant, 15 preeclamptic, and 10 healthy pregnant women and assayed for free gravidin by enzyme-linked immunosorbent assay. Neutrophil phospholipase A 2 and respiratory burst activities were determined in the presence of isolated free gravidin by cellular arachidonic acid release and superoxide anion production. Results: Levels of free gravidin were higher in the healthy pregnant (36.1 ± 5.5 ng/mL) and preeclamptic (17.8 ± 2.8 ng/mL) groups than in the nonpregnant control group (3.9 ± 0.5 ng/mL) and were significantly different between pregnancy groups ( P < .01, Mann-Whitney U test). Free gravidin caused a concentration dependent decrease in N -formyl-methionyl-leucyl-phenylalanine–stimulated neutrophil arachidonic acid release (inhibitory concentration of 50% 25 nmol/L) and superoxide anion generation (inhibitory concentration of 50% 32 nmol/L). Conclusions: Circulating levels of free gravidin are reduced in pre-eclampsia compared with normal pregnancy. This may encourage an increase in the respiratory burst of neutrophils in pre-eclampsia and could contribute to the oxidative stress and vascular damage that characterize this disease. (Am J Obstet Gynecol 1998;179:1305-11.)

Neutrophil arachidonic acid level and adhesive capability are increased in essential hypertension.

We recently demonstrated that arachidonic:linoleic acid ratio of erythrocytes of essential hypertension patients is greater than normal. To investigate fatty acid composition, capability for adhesion to biological substrate and expression of beta2 integrins of leucocytes obtained from peripheral blood and skin window exudate of essential hypertension patients. Neutrophil activation state was evaluated by reproducing the various conditions occurring in vivo during the life of the cell (i.e. under the 'resting' condition, such as in peripheral blood, and 'primed' condition, such as after transmigration through the endothelium and after administration of specific chemo-attractants). Because both peripheral blood and skin window leucocytes of the subjects were obtained on the same day, we could be sure that there had been no dietary influences on changes in levels of fatty acid. Thus, the observed changes should reliably reflect the metabolic rate of utilization of fatty acids coupled to the activation and migration of cells. Leucocytes from essential hypertension patients were richer in arachidonic acid than were the corresponding cells from normotensive subjects; this difference was also evident for functionally activated skin window leucocytes, in spite of there having been a greater loss of poly-unsaturated fatty acids and arachidonic acid after migration. Moreover, a greater than normal arachidonic acid:linoleic acid ratio was shown for the first time to apply for leucocytes of essential hypertension patients, so extending our previous findings on the erythrocytes. Leucocytes from essential hypertension patients, collected both from peripheral blood and from skin window exudate, proved far more adhesive than the corresponding cells from age-matched and sex-matched controls, but this was not associated with a quantitative hyperexpression of beta2 integrins. The results suggest that an increase in availability of arachidonic acid in leucocytes could be a further expression of the generalized disturbance of fatty acid levels associated with essential hypertension and that a condition of hyperadhesion of neutrophils could occur spontaneously in vivo during the course of hypertension.

Metabolism of arachidonic acid in neutrophils in patients with melanoma

Metabolism of arachidonic acid in neutrophils in patients with melanoma

The discovery of LY293111, a novel, potent and orally active leukotriene B4 receptor antagonist of the biphenylphenol class.

Leukotriene B4 (LTB4) has been implicated as a key pro-inflammatory mediator in human diseases such as asthma, inflammatory bowel disease, psoriasis and arthritis (1). LTB4’s role in human disease is supported by the fact that via G-protein coupled receptor mediated events, this product of the 5-lipoxygenase pathway of arachidonic acid metabolism initiates granulocyte chemotaxis, aggregation, upregulation of adhesion molecules and the release of Superoxide and degradative enzymes (2). The existence of elevated concentrations of LTB4 in tissues or interstitial fluids of patients with inflammatory diseases also supports the role of LTB4 as a pro-inflammatory mediator (1). However, the presence of LTB4 associated with the clinical manifestations of these inflammatory diseases only indirectly implicates it as a major player in inflammatory processes. The delineation of LTB4’s role in inflammation requires the development of potent and selective LTB4 receptor antagonists.KeywordsInflammatory Bowel DiseaseReceptor BindingHuman NeutrophilArachidonic Acid MetabolismBioactive LipidThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Identification and molecular characterization of the CalB domain of the cytosolic phospholipase A2 (cPLA2) in human neutrophils.

The receptor-mediated activation of human neutrophils by ligands such as chemotactic peptide N-formyl-methionine-leucine-phenylalanine (fMLP) is coupled with the activation of phospholipases C and A2 (1,2). Phospholipase A2 (PLA2) hydrolyzes arachidonic acid (AA) and lysophospholipid from the sn-2 position of 1-alkyl-2-arachidonoyl-sn-3-glycerophosphocholine of the membrane (3). Currently, it is accepted that the cytosolic PLA2 (cPLA2) and not the secretory PLA2 (sPLA2) is primarily responsible for the release of AA (4) in most of the cell systems. The reasons for this assumption are based on the observations such as low requirements of Ca++ concentrations (micromolar for cPLA2 vs milimolar for sPLA2), fatty acid preference of cPLA2 at the sn-2 position (5), resistance of cPLA2 to disulfide-reducing agents (4) and increased concentrations of cytosolic Ca++ (5,6). In addition, it is stimulated by phosphorylation through protein kinase C (PKC), but inhibited by staurosporine, an inhibitor of PKC (7). Conversely, previous studies from our laboratory have shown that the inhibition of PKC by staurosporine potentiated the activation of PLC and PLA2 in fMLP-challenged human PMN (8–10).KeywordsArachidonic AcidHuman NeutrophilArachidonic Acid ReleaseU937 Cell LineCytosolic PhospholipaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Calcium mobilization and right-angle light scatter responses to 12-oxo-derivatives of arachidonic acid in neutrophils: evidence for the involvement of the leukotriene B 4 receptor

The biological activities of two carbonyl compounds derived from arachidonic acid, (5 Z,8 Z,10 E,14 Z)-12-keto-5,8,10,14-eicosatetraenoic acid (12-OxoETE) and (5 Z,8 Z,10 E)-12-oxo-5,8,10-dodecatrienoic acid (12-OxoDTrE) were investigated. The ability of these compounds to induced a mobilization of calcium and to trigger a right-angle scatter response in isolated peripheral blood human neutrophils was determined. The two compounds induced a rapid and dose-dependent increase in the concentration of cytoplasmic free calcium; these effects were clearly detectable at concentrations ⩾10 −8 M. Pre-exposure of neutrophils to leukotriene B 4 completely abolished the calcium mobilization induced by 12-OxoDTre and 12-OxoETE, while pre-exposure of the cells to the carbonyl compounds only slightly reduced the response to subsequent stimulation of neutrophils by leukotriene B 4.The carbonyl compounds also induced a decrease in right-angle light scatter and these effects were abolished by pretreatment of neutrophils with leukotriene B 4. These data demonstrate that 12-OxoETE and 12-OxoDTrE show significant agonist activities towards human neutrophils and strongly suggest that their mechanisms of action involve the leukotriene B 4 binding sites or a common activation sequence.

Metabolism of arachidonic acid in neutrophils from alloxan-diabetic rabbits

The production of 5-lipoxygenase products from arachidonic acid was investigated in polymorphonuclear leukocytes (PMNL) isolated from non-diabetic and alloxan-induced diabetic rabbits: (i) production of 5-hydroxyeicosatetraenoic acid, leukotriene B 4, and the two 6-trans-leukotriene B 4 isomers were significantly decreased in the PMNL of diabetic rabbits when compared to non-diabetic rabbits; (ii) production of LTB 4 and 5-HETE from diabetic PMNL required the addition of Ca 2+ and A23187 to a greater degree than control incubations; and (iii) the availability of substrate in the PMNL of diabetics was not a limiting factor for 5-lipoxygenase product formation. Alternative pathways of arachidonic acid metabolism were also evaluated: the recovery of exogenous leukotriene B 4 and 5-hydroxyeicosatetraenoic acid were identical using PMNL from control and diabetic rabbits and peptido-leukotrienes were not detected by radioimmunoassay. The data suggest that the activity of 5-lipoxygenase and the production of 5-hydroperoxyeicosatetraenoic acid in the diabetic PMNL may be limiting factors since the formation of leukotriene B 4, leukotriene B 4 isomers, and 5-hydroxyeicosatetraenoic acid are depressed in PMNL of diabetic rabbits. Alternative pathways do not account for the conversion of arachidonic acid to other products nor are the elimination pathways for LTB 4 and 5-HETE different. Decreased formation of 5-hydroxyeicosatetraenoic acid and leukotriene B 4 could predispose diabetic subjects to infection due to a decrease in mediators leading to the local accumulation of PMNL in the inflammatory response.

Evaluation of Tenidap (CP-66,248) on human neutrophil arachidonic acid metabolism, chemotactic potential and clinical efficacy in the treatment of rheumatoid arthritis

Ten patients with rheumatoid arthritis (RA) were evaluated in a placebo-controlled, double-blind study examining the clinical efficacy of a novel nonsteroidal anti-inflammatory agent: Tenidap (CP-66,248). RA patients receiving active drug therapy (n = 6) demonstrated clinically significant improvements in observer assessment of pain (p less than 0.025), painful joint count (p less than 0.010), and overall clinical assessment as based on a modified rheumatoid activity index, MRAI (p less than 0.025). In parallel laboratory assays, Tenidap was found to exhibit a significant in vitro dose-dependent inhibition of ionophore-stimulated neutrophil production of the 5-lipoxygenase product: [3H]leukotriene B4 (LTB4). Although more importantly, Tenidap was also found to exhibit an in vitro dose-dependent inhibition (IC50 20 microM) of the ionophore-stimulated release (deacylation) of the precursor [3H]arachidonic acid (AA) from membrane phospholipids. In further studies, Tenidap did not have any effect on fMLP-induced neutrophil chemotactic response. These results suggest that one of the possible mechanisms for the clinical effectiveness of this agent, may be through its effect at inhibiting the release of free AA from membrane phospholipids and therefore limiting its further metabolism into certain biologically-active inflammatory lipids.

Cytokine- and calcium ionophore A23187-mediated arachidonic acid metabolism in neutrophils

Arachidonic acid (AA) metabolism is implicated as an intracellular and/or intercellular second messenger system for the transmission of cytokine-initiated signals that affect neutrophils and mediate systemic toxicity. The purpose of the present study is to ascertain if cytokines that are known to affect neutrophil function in vivo and in vitro directly stimulate neutrophil AA metabolism in vitro. The recombinant human cytokines multi-colony stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 1, tumor necrosis factor (TNF), and interleukin 6 and the calcium ionophore A23187 were incubated with purified 14C-AA radiolabeled human peripheral blood neutrophils and the effects were assayed by one- and two-dimensional thin layer lipid chromatography. None of the cytokines appeared to induce the release of cell-incorporated AA or to increase the level of radiolabeled phosphatidic acid. TNF induces severe systemic toxicity that is inhibited by cyclooxygenase inhibitors, which suggests a role for AA metabolites in the pathophysiologic effects of TNF; we have confirmed that TNF and endotoxin act synergistically to induce indomethacin-inhibitable fatal shock in rats. However, when in 3H-AA radiolabeled human neutrophils were incubated with TNF in kinetic, cold-chase, and TNF preincubation experiments, TNF was not found to increase AA metabolism, although changes in the intracellular neutral lipid content were noted. GM-CSF, which has been reported by previous investigators to directly induce the release of AA, did not release neutrophil-associated 3H-AA. In conclusion, the direct release of AA from membrane-associated phospholipids does not appear to be a major second messenger pathway for cytokine-initiated activation of neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

Human granulocyte-macrophage colony-stimulating factor and other cytokines prime human neutrophils for enhanced arachidonic acid release and leukotriene B4 synthesis.

Human recombinant granulocyte-macrophage CSF (GM-CSF) "primes" neutrophils for enhanced biologic responses to a number of secondary stimuli. Here, we examined the properties of neutrophil priming by GM-CSF and other growth factors such as human rTNF and granulocyte CSF. Although GM-CSF has a negligible direct effect on [3H]arachidonic acid release, it enhances or "primes" neutrophils for three- to fivefold increased release of [3H]arachidonic acid, induced by 1.0 microM A23187 and the chemotactants FMLP, platelet-activating factor, and leukotriene B4 (LTB4) (all 0.1 microM). The priming effects of GM-CSF were concentration- and time-dependent (maximum 100 pM, 1 h at 23 degrees C), and consistent with the determined dissociation constant of the human GM-CSF receptor. Indomethacin (10(-8) M), cycloheximide (100 micrograms/ml), and pertussis toxin (200 ng/ml, 2 h at 37 degrees C) had no effect on GM-CSF-, A23187, or platelet-activating factor-induced [3H]arachidonic acid release. The lipoxygenase inhibitor, nordihydroguaiaretic acid, however, totally abolished A23187-induced [3H]arachidonic acid release from both diluent- and GM-CSF-treated neutrophils. Consistent with this observation, we found that GM-CSF-pretreated neutrophils synthesize increased levels of LTB4 after stimulation with A23187 and chemotactic factors. GM-CSF enhances neutrophil arachidonic acid release and LTB4 synthesis, and thereby may amplify the inflammatory response to chemotactic factors and other physiologically relevant stimuli.

1- [formula omitted]-glycero-3-phosphocholine: A novel source of arachidonic acid in neutrophils stimulated by the calcium ionophore A23187

Rabbit peritoneal neutrophils incorporated [ 14C]arachidonic acid into seven molecular species of choline-containing phosphoglycerides. These 2-[ 14C]arachidonoyl species differed with respect to the alkyl ether or acyl residue bound at the sn -1 position; four of the seven were ether-linked. Stimulation with calcium ionophore A23187 induced a proportionate release of arachidonate from all seven molecular species: 40% of the released arachidonate came from alkyl ether species. Thus, 1- O -alkyl-2-arachidonoyl- sn -glycero-3-phosphocholine (GPC) is a significant source of metabolizable arachidonic acid. Since 1- O -alkyl-2-lyso-GPC is the metabolic precussor of platelet activating factor, these results further interrelate pathways forming arachidonate metabolites and platelet activating factor; they also supply a rationale for the observation that both classes of stimuli form concomitantly during cell activation.

Parallel inhibition of neutrophil arachidonic acid metabolism and lysosomal enzyme secretion by nordihydroguaiaretic acid

Arachidonic acid at concentrations from 0.2 to 2.0 × 10− 6M induces the secretion of lysosomal enzymes from cytochalasin B-treated rabbit neutrophils. These concentrations of arachidonic acid are metabolized primarily to hydroxyeicosatetraenic acids rather than to cyclooxygenase products. A good correlation is observed between the extent of arachidonic acid metabolism and the secretion of lysosomal enzymes. Nordihydroguaiaretic acid (1−10μM) inhibits both lysosomal enzyme secretion and the production of lipoxygenase products by neutrophils.