Pierre Borgeat

Comparative effects of indomethacin, acetylenic acids, 15-HETE, nordihydroguaiaretic acid and BW755C on the metabolism of arachidonic acid in human leukocytes and platelets.

Human leukocytes and platelets were preincubated with inhibitors of the oxidative metabolism of arachidonic acid, viz, indomethacin, BW755C, 5, 8, 11, 14-eicosatetraynoic acid (ETYA), 5, 8, 11-eicosatriynoic acid, 15S-hydroxy-5, 8, 11, 13-(Z, Z, Z, E)-eicosatetraenoic acid (15-HETE) and nordihydroguaiaretic acid (NDGA), prior to stimulation with the ionophore A23187 in the presence or absence of exogenous arachidonic acid. Eleven metabolites of arachidonic acid derived from the cyclooxygenase and the 5-, 12- and 15-lipoxygenases were measured by reversed-phase high pressure liquid chromatography (RP-HPLC). Indomethacin was the best inhibitor of the platelet cyclooxygenase (ID50 less than 10(-7)M) as assessed by measurement of 12-hydroxy-heptadecatrienoic acid (HHT); ETYA was the most potent inhibitor of the 12- and 15-lipoxygenases (ID50 approximately 3 X 10(-7)M) whereas NDGA was the most potent and selective inhibitor of the 5-lipoxygenase (ID50 approximately 3 X 10(-7)M).

Direct demonstration of increased intracellular concentration of free calcium in rabbit and human neutrophils following stimulation by chemotactic factor

An increase in the level of intracellular free calcium concentration in rabbit and human neutrophils stimulated by chemotactic factors has been demonstrated directly using the calcium-sensitive fluorescent probe quin-2. Addition of f-Met-Leu-Phe (10(-9) M), C5a (3 x 10(-9) M) or leukotriene B4 (6 x 10(-8) M) to the neutrophils induces a rapid increase in the intracellular concentration of free calcium that reaches a maximum value 15 seconds following stimulation. At concentrations of f-Met-Leu-Phe less than 10(-8) M the enhancement is dose dependent with an ED50 of 8 x 10(-11) M and is significantly reduced in the presence of EGTA in the suspending medium.

Inhibition of adenosine 3′,5′-monophosphate accumulation in anterior pituitary gland in vitro by growth hormone-release inhibiting hormone

Summary 1 × 10 −7 M growth hormone-release inhibiting hormone (GH-RIH or somatostatin) leads to a 50% inhibition of adenosine 3′,5′-monophosphate (cyclic AMP) accumulation in anterior pituitary tissue during the first 2 min of incubation. This lowering of cyclic AMP levels is accompanied by inhibition of the release of immunoreactive growth hormone and thyrotropin. GH-RIH has also a marked inhibitory effect on the theophyline- and prostaglandin E 2 -induced accumulation of adenohypophyseal cyclic AMP.

Intraperitoneal injection of zymosan in mice induces pain, inflammation and the synthesis of peptidoleukotrienes and prostaglandin E2

Intraperitoneal injection of zymosan in mice induced rapid extravasation and accumulation of plasma protein in the peritoneal cavity. Neutrophils began to appear in the peritoneal cavity after a lag period of approximately 3 hours. The injected mice exhibited a pain response (writhing) during the first 30 minutes after injection, but writhing ceased before protein or cell accumulation had reached maximum levels. The injection of zymosan induced synthesis of PGE2 (measured by RIA) which reached maximum levels at 30 minutes, then declined slowly. Peptido-leukotriene levels (detected by bioassay, RIA and HPLC) increased rapidly after injection, reached a peak within an hour of injection and declined to undetectable levels within 4 hours. The early peptido-LT was predominantly LTC4, while later, LTE4 was the major component. LTD4 levels remained low throughout and no LTB4 was detected at any time. Indomethacin treatment elevated levels of peptido-LTs, reduced PGE2 levels and inhibited writhing. Phenidone reduced peptido-LT levels. In vitro studies demonstrated that zymosan stimulates LTC4 synthesis by peritoneal cells whereas LTE4, LTD4, LTB4 or monoHETES were not detectable (using HPLC methods). The source of enzymes responsible for the in vivo metabolism of LTC4 to LTD4 and LTE4 could not be identified.

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.

Expression and activity of prostaglandin endoperoxide synthase-2 in agonist-activated human neutrophils

Proinflammatory agents were assessed for their capacity to stimulate the expression of the inducible cyclooxygenase isoform (COX‐2) in human neutrophils. A number of agents, including PMA, opsonized bacteria and zymosan, LPS, GM‐CSF, TNF‐α, and fMLP, induced COX‐2 protein expression through signaling pathways involving transcription and protein synthesis events. Northern blots showed that freshly isolated neutrophils expressed low levels of COX‐2 mRNA, which rapidly increased after incubation with inflammatory agents. A characterization of the signal transduction pathways leading to COX‐2 protein expression was initiated. In LPS‐treated neutrophils, efficient induction of COX‐2 required the presence of serum and involved ligand binding to the CD14 surface antigen. The specific inhibitor of p38 mitogen‐activated protein kinase (p38 MAPK), SB 203580, had little effect on the induction of COX‐2 expression in neutrophils, in contrast to what had been previously observed with other inflammatory cell types. Depending on the agonist present, ethanol differentially blocked the stimulated expression of COX‐2, raising the possibility that phospholipase D activation might take part in the process of COX‐2 induction. Major COX‐2‐derived prostanoids synthesized by inflammatory neutrophils were identified by liquid‐chromatography and tandem mass‐spectrometry as TXA2 and PGE2. The agonist‐induced synthesis of TXA2 and PGE2 was effectively blocked by cycloheximide and by the specific COX‐2 inhibitor NS‐398. These results show that COX‐2 can be induced in an active state by different classes of inflammatory mediators in the neutrophil. They support the concept that, in these cells, the COX‐2 isoform is preeminent over COX‐1 for the stimulated‐production of prostanoids, and also suggest that neutrophil COX‐2 displays a distinct profile of expression among circulatory cells.—Pouliot, M., Gilbert, C., Borgeat, P., Poubelle, P. E., Bourgoin, S., Créminon, C., Maclouf, J., McColl, S. R., Naccache, P. H. Expression and activity of prostaglandin endoperoxide synthase‐2 in agonist‐activated human neutrophils. FASEB J. 612, 1109–1123 (1998)

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.

A Functional Toll-Like Receptor 8 Variant Is Associated with HIV Disease Restriction

BACKGROUND Toll-like receptors (TLRs) play an important role in the innate immune response to pathogens. TLR8 has been found to recognize RNA derived from various viruses, including human immunodeficiency virus (HIV). Presently, very little is known about the influence of TLR8 genetic variation on susceptibility to and progression of HIV disease. METHODS AND RESULTS We genotyped a population of 782 HIV-positive adults and 550 healthy control subjects for 3 nonsynonymous TLR8 single-nucleotide polymorphisms. We found that the presence of the most frequent TLR8 polymorphism, TLR8 A1G (rs3764880), confers a significantly protective effect regarding progression of the disease. In overexpression assays, we demonstrated that this receptor variant displays impaired NF-kappaB activation in vitro. Furthermore, we analyzed different cell types obtained from individuals differing in their TLR8 genotype and assessed their response to TLR8 ligands in vitro. The presence of the mutated receptor variant was associated with modulation of cytokine secretion profiles and lipid mediator synthesis patterns in monocytes and neutrophils. CONCLUSIONS This first report of a functional TLR8 variant associated with a different clinical course of an RNA viral disease may have implications for the individual risk assessment of patients infected with HIV and other RNA viruses as well as for future HIV vaccine development.

The action of leukotriene B4 (LTB4) on the lung

The actions of leukotriene B4 (LTB4), a member of a newly discovered pathway of metabolism of arachidonic acid, were investigated both on the guinea-pig perfused lung preparation and on the parenchymal strip and compared to histamine and Slow Reacting Substance of Anaphylaxis (SRS-A). LTB was prepared from human polymorphonuclear leukocytes, extracted and purified by chromatography (Silicic acid and HPLC) and its purity was determined by gas chromatography and mass spectrometry. LTB4 is three times more potent than histamine (molar concentration) to contract the parenchymal strips and the contraction to LTB4 as well as to SRS-A lasted longer. The contraction to LTB4 is blocked by indomethacin (20 micrograms/ml), reduced by polyphloretin phosphate (50 micrograms/ml) and unaffected by FPL-55712 (1 micrograms/ml). Following its injection in the pulmonary artery of a perfused lung, LTB4 (1 microgram) induced the release of RCS (Rabbit Aorta Contracting Substance: a mixture of prostaglandins and thromboxanes) which can be abolished by indomethacin (1 microgram/ml). These findings suggest (a) that in the lung, LTB4 is a myotropic agent three times more powerful than histamine (b) that LTB4 stimulated a receptor which is different of histamine of SRS-A receptors, and (c) that its contractile action in the lung is mediated by prostaglandins and thromboxanes.