Serge Picard

Transformation of arachidonic acid in leukocytes. Isolation and structural analysis of a novel dihydroxy derivative.

Abstract A suspension of mixed peripheral blood leukocytes was incubated with arachidonic acid. After ether extraction and silicic acid fractionation of the products, the fraction containing the mono- and dihydroxy derivatives of arachidonic acid was further analyzed by high pressure liquid chromatography on silica gel and octadecyl silica (reversed-phase) columns. A previously undescribed metabolite was detected and isolated in pure form. The compound co-chromatographed with leukotriene B4 on octadecyl silica but was eluted earlier than leukotriene B4 from silica gel columns. Ultraviolet spectrophotometry, gas chromatography-mass spectrometry, ozonolysis and steric analysis indicated that the new metabolite was the 5S,12S-dihydroxy-6,8,10,14-icosatetraenoic acid. The yield of the novel dihydroxy acid was 1 to 4% of the added substrate. The new metabolite showed less than 1% of the myotropic activity of leukotriene B4 on the guinea pig lung parenchymal strip.

Eosinophil-rich human polymorphonuclear leukocyte preparations characteristically release leukotriene C4 on ionophore A23187 challenge

Blood samples were obtained from a group of 20 patients with hypereosinophilia (greater than or equal to 1500 eosinophils/mm3). The polymorphonuclear leukocytes (PMNLs) were prepared from blood treated with ethylenediaminetetra-acetic acid by successive dextran sedimentation of the red blood cells, separation of mononuclear leukocytes and PMNLs on Ficoll-Paque, and ammonium chloride treatment of the PMNL fraction. The eosinophil content of the final PMNL preparations ranged from 15% to 75%, as assessed by Wright-stained smears, and the remaining leukocytes were predominantly neutrophils with only 3% to 5% mononuclear cells. The eosinophil-rich PMNL preparations as well as PMNL preparations from normal volunteers were incubated under various conditions and the arachidonic acid metabolites were analyzed by reverse-phase high-performance liquid chromatography. The synthesis of 5-lipoxygenase products was strongly stimulated by the ionophore A23187 in both normal and eosinophil-rich PMNL preparations. Whereas the normal PMNL preparations, which were eosinophil poor, produced 10 to 25 times more leukotriene B4 than leukotriene C4, the eosinophil-rich PMNL preparations characteristically released leukotriene C4 in equal or up to 20 times greater amounts than leukotriene B4.

Pharmacological activity of leukotrienes A4, B4, C4 and D4 on selected guinea-pig, rat, rabbit and human smooth muscles

The myotropic activity of leukotrienes A4, B4, C4, D4 and histamine has been evaluated on selected smooth muscle preparations. LTA4, B4, C4 and D4 were several times more potent than histamine on the guinea-pig lung parenchymal strip, while on the guinea-pig trachea, LTB4 was less active. The guinea-pig ileum either in segments or in strips of longitudinal muscles responses well to LTC4, LTD4 and histamine but not to LTA4 and LTB4. Rat and rabbit lung parenchymal strip showed very little sensitivity for leukotrienes whereas human parenchymal strips and bronchi were nearly as sensitive as the guinea-pig lung.

Studies on the mechanism of formation of the 5S,12S-dihydroxy-6,8,10,14(E,Z,E,Z)-icosatetraenoic acid in leukocytes

Incubation of peripheral blood leukocytes with arachidonic acid (and ionophore A23187) led to the formation of leukotriene B4, delta 6-trans-leukotriene B4, delta 6-trans-12-epi-leukotriene B4, 5-hydroxy-icosatetraenoic acid, 12-hydroxy-icosatetraenoic acid and of 5S,12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-icosatetraenoic acid (5S,12S-DiHETE). Incubation of leukocytes with leukotriene A4 resulted in the formation of leukotriene B4 and of its two delta 6-trans-isomers but not of the 5S, 12S-DiHETE. 18O2 labeling experiments have shown that the hydroxyl groups at C5 and C12 in the 5S,12S-DiHETE are derived from molecular oxygen. The tetraacetylenic analog of arachidonic acid was found to be potent inhibitor of the formation of the 5S,12S-DiHETE whereas it potentiated the synthesis of the 5-hydroxy acid and of leukotriene B4. Addition of the 12-hydroxy-icosatetraenoic acid to leukocytes, or of the 5-hydroxy-icosatetraenoic acid to a suspension of platelets caused the formation of the 5S,12S-DiHETE. It is concluded that the 5S,12S-DiHETE is not derived from leukotriene A4 but is a product of the successive reactions of arachidonic acid with two lipoxygenases of different positional specificities.

Mechanisms of the priming effect of lipopolysaccharides on the biosynthesis of leukotriene B4 in chemotactic peptide-stimulated human neutrophils

The goal of this study was to explain the priming effect of lipopolysaccharides (LPS) in human polymorphonuclear leukocytes on leukotriene B4 (LTB4) biosynthesis after stimulation with the receptor‐mediated agonist formyl‐methionyl‐leucylphenylalanine (fMLP). This priming effect for LTB4 biosynthesis was maximal after a 30 min preincubation with LPS but was lost when incubations were extended to 90 min or longer. Priming with LPS resulted in an enhanced maximal activation of 5‐lipoxygenase (5‐ to 15‐fold above unprimed cells) as well as a prolonged activation of the enzyme after stimulation with fMLP compared to that measured in unprimed cells. The activation of 5‐lipoxygenase was associated with its translocation to the nuclear fraction of the cell after stimulation of LPS‐primed cells but not of unprimed cells. Priming of cells with LPS also resulted in an enhanced capacity (fivefold increase) for arachidonic acid (AA) release after stimulation with fMLP compared to unprimed cells as measured by mass spectrometry. This release of AA was very efficiently blocked in a dose‐dependent manner by the 85 kDa cytosolic phospholipase A2 (PLA2) inhibitor MAFP (IC50 = 10nM) but not by the 14 kDa secretory PLA2 inhibitor SB 203347 (up to 5 µM), indicating that the 85 kDa cPLA2 is the PLA2 responsible for AA release in response to receptor‐mediated agonists. In accord with inhibitor studies, the LPS‐mediated phosphorylation of cPLA2 followed the same kinetics as the priming for AA release, and a measurable fMLP‐induced translocation of cPLA2 was observed only in primed cells. As with AA release and LTB4 biosynthesis, both the phosphorylation and capacity to translocate cPLA2 were reversed when the preincubation period with LPS was extended to 120 min. These results explain some of the cellular events responsible for the potentiation and subsequent decline of functional responses of human polymorphonuclear leukocytes recruited to inflammatory foci.—Surette, M. E., Dallaire, N., Jean, N., Picard, S., Borgeat, P. Mechanisms of the priming effect of lipopolysaccharides on the biosynthesis of leukotriene B4 in chemotactic peptide‐stimulated human neutrophils. FASEB J. 12, 1521–1531 (1998)

The Toll-like receptor 7/8-ligand resiquimod (R-848) primes human neutrophils for leukotriene B4, prostaglandin E2 and platelet-activating factor biosynthesis

Toll‐like receptors (TLR) recognize pathogen‐associated molecular patterns and play important roles in the innate immune system. While single‐stranded viral RNA is the natural ligand of TLR7/TLR8, the imidazoquinoline resiquimod (R‐848) is recognized as a potent synthetic agonist of TLR7/TLR8. We investigated the effects of TLR7/8 activation on lipid mediator production in polymorphonuclear leukocytes exposed to R‐848. Although R‐848 had minimal effects by itself, it strongly enhanced leukotriene B4 formation on subsequent stimulation by fMLP, platelet‐activating factor, and the ionophore A23187. R‐848 acted via TLR8 but not TLR7 as shown by the lack of effect of the TLR7‐specific ligand imiquimod. Priming with R‐848 also resulted in enhanced arachidonic acid release and platelet‐activating factor formation following fMLP stimulation, as well as enhanced prostaglandin E2 synthesis following the addition of arachidonic acid. Western blot analysis demonstrated that R‐848 induced the phosphorylation of the cytosolic phospholipase A2α, promoted 5‐lipoxygenase translocation and potently stimulated the expression of the type 2 cyclooxygenase. Bafilomycin A1, an inhibitor of endosomal acidification, efficiently inhibited all R‐848‐induced effects. These studies demonstrate that TLR8 signaling strongly promotes inflammatory lipid mediator biosynthesis and provide novel insights on innate immune response to viral infections.—Hattermann K., Picard, S., Borgeat, M., Leclerc, P., Pouliot, M., Borgeat P. The Toll‐like receptor 7/8‐ligand re‐siquimod (R‐848) primes human neutrophils for leuko‐triene B4, prostaglandin E2 and platelet‐activating factor biosynthesis. FASEB J. 21, 1575–1585 (2007)

Arachidonic Acid Regulates the Translocation of 5-Lipoxygenase to the Nuclear Membranes in Human Neutrophils

Elevation of the intracellular cAMP concentration in agonist-activated human neutrophils (PMN) leads to the concomitant inhibitions of arachidonic acid (AA) release, 5-lipoxygenase (5-LO) translocation, and leukotriene (LT) biosynthesis. We report herein that exogenous AA completely prevents cAMP-dependent inhibition of 5-LO translocation and LT biosynthesis in agonist-activated PMN. Moreover, the group IVA phospholipase A2 inhibitor pyrrophenone and the MEK inhibitor U-0126 inhibited AA release and 5-LO translocation in activated PMN, and these effects were also prevented by exogenous AA, demonstrating a functional link between AA release and 5-LO translocation. Polyunsaturated fatty acids of the C18 and C20 series containing at least three double bonds located from carbon 9 (or closer to the carboxyl group) were equally effective as AA in restoring 5-LO translocation in pyrrophenone-treated agonist-activated PMN. Importantly, experiments with the 5-LO-activating protein inhibitor MK-0591 and the intracellular Ca2+ chelator BAPTA-AM demonstrated that the AA-regulated 5-LO translocation is FLAP- and Ca2+-dependent. Finally, the redox and competitive 5-LO inhibitors L-685,015, L-739,010, and L-702,539 (but not cyclooxygenase inhibitors) efficiently substituted for AA to reverse the pyrrophenone inhibition of 5-LO translocation, indicating that the site of regulation of 5-LO translocation by AA is at or in the vicinity of the catalytic site. This report demonstrates that AA regulates the translocation of 5-LO in human PMN and unravels a novel mechanism of the cAMP-mediated inhibition of LT biosynthesis.

Extra Domain A of Fibronectin Primes Leukotriene Biosynthesis and Stimulates Neutrophil Migration Through Activation of Toll-like Receptor 4

OBJECTIVE There is increasing evidence of a role for Toll-like receptors (TLRs) in inflammatory arthritis. The extra domain A (ED-A)-containing isoform of fibronectin is generated under pathologic conditions such as rheumatoid arthritis, and ED-A has been identified as an endogenous TLR-4 ligand. Leukotriene B4 (LTB4) and polymorphonuclear neutrophils (PMNs) play a critical role in murine models of inflammatory arthritis. The aim of this study was therefore to investigate the putative effects of ED-A on leukotriene biosynthesis and PMN migration through TLR signaling. METHODS The effect of recombinant human ED-A (rhED-A) on leukotriene biosynthesis was evaluated in isolated human blood PMNs and monocytes by high-performance liquid chromatography. The capacity of rhED-A to stimulate PMN migration was evaluated using a transendothelial/matrix migration assay in vitro and the mouse air-pouch model in vivo. RESULTS Recombinant human ED-A efficiently primed the biosynthesis of LTB4 in PMN and monocyte suspensions. This priming effect was dependent on TLR-4 activation, since the TLR-4-signaling inhibitor CLI-095 completely blocked the effect of rhED-A but not that of other TLR ligands (R-848, Pam2 CSK4) or cytokines. Moreover, rhED-A stimulated transendothelial migration of PMNs in vitro, which was inhibited by 50-60% with the LTB4 receptor 1 (BLT1) antagonist CP105,696 or the cytosolic phospholipase A2 α inhibitor pyrrophenone. In vivo, rhED-A induced a significant PMN recruitment into the air pouch of C3H/HeOuJ mice (expressing functional TLR-4), but not in C3H/HeJ mice (expressing nonsignaling TLR-4). CONCLUSION These results demonstrate the ability of rhED-A to promote LTB4 biosynthesis and PMN migration through TLR-4 activation, thus providing new insights on TLR-dependent mechanisms of regulation of LTB4 biosynthesis and PMN infiltration in inflammatory joint diseases.

Potentiation of neutrophil cyclooxygenase-2 by adenosine: an early anti-inflammatory signal

Neutrophils, which are often the first to migrate at inflamed sites, can generate leukotriene B4 from the 5-lipoxygenase pathway and prostaglandin E2 through the inducible cyclooxygenase-2 pathway. Adenosine, an endogenous autacoid with several anti-inflammatory properties, blocks the synthesis of leukotriene B4 while it potentiates the cyclooxygenase-2 pathway in fMLP-treated neutrophils, following activation of the A2A receptor. Using the murine air pouch model of inflammation, we observed that inflammatory leukocytes from mice lacking the A2A receptor have less cyclooxygenase-2 induction than wild-type animals. In human leukocytes, A2A receptor activation specifically elicited potentiation of cyclooxygenase-2 in neutrophils, but not in monocytes. Signal transduction studies indicated that the cAMP, ERK1/2, PI-3K and p38K intracellular pathways are implicated both in the direct upregulation of cyclooxygenase-2 and in its potentiation. Together, these results indicate that neutrophils are particularly important mediators of adenosines effects. Given the uncontrolled inflammatory phenotype observed in knockout mice and in view of the potent inhibitory actions of prostaglandin E2 on inflammatory cells, an increased cyclooxygenase-2 expression resulting from A2A receptor activation, observed particularly in neutrophils, may take part in an early modulatory mechanism promoting anti-inflammatory activities of adenosine.

Toll-like receptor ligands induce polymorphonuclear leukocyte migration: key roles for leukotriene B4 and platelet-activating factor

Activation of toll‐like receptors (TLRs) and polymorphonuclear leukocyte (PMN) accumulation at infection sites are critical events of host defense. The involvement of leukotriene (LT) B4 and platelet‐activating factor (PAF) in TLR ligand‐induced activation of inflammatory cell functions is essentially unknown. Using an in vitro model of human PMN migration through human endothelial cell monolayers’ we demonstrate that prototypic ligands of TLR1/2, 2/6, 3, 4, 5, and 7/8 promote PMN migration, an effect markedly inhibited by 3 LTB4 receptor antagonists (70–80% inhibition at 100 nM compared to vehicle‐treated cells), 3 PAF receptor antagonists (20–50% inhibition at 10 nM), 3 LT biosynthesis inhibitors (75–85% inhibition at 100 nM), and 1 cytosolic phospholipase A2α (cPLA2α) inhibitor (90% inhibition at 1 μM). Accordingly, selected TLR ligands caused Ser505‐phosphorylation of cPLA2α and measurable LTB4 and PAF biosynthesis in the transmigration assay. As negative controls, interleukin‐8‐ and formyl‐methionyl‐leucyl‐phenylalanine‐elicited migration in vitro was not inhibited either by an LTB4 receptor antagonist or by the cPLA2α inhibitor. Finally, LTB4 and PAF receptor antagonists inhibited (up to ~65% at optimal doses) TLR ligand‐induced PMN infiltration in the mouse air‐pouch model. These studies unravel the critical involvement of de novo LTB4 and PAF biosynthesis in PMN migration elicited by TLR ligands.—Lefebvre, J. S., Marleau, S., Milot, V., Lévesque, T., Picard, S., Flamand, N., Borgeat, P. Toll‐like receptor ligands induce polymorphonuclear leukocyte migration: key roles for leukotriene B4 and platelet‐activating factor. FASEB J. 24, 637–647 (2010). www.fasebj.org