5-Lipoxygenase pathway-derived products of arachidonic acid released by human eosinophils activated in vitro have been measured by using radioimmunoassays specific for leukotriene B4 (LTB4) and for sulfidopeptide leukotrienes including leukotriene C4 (LTC4). Eosinophil-enriched leukocytes (mean, 85% eosinophils) from five hypereosinophilic donors activated with 5.0 pAM ionophore A23187 for 15 min at 37C in the presence of 50 mM L-serine released 69 ± 28 and 1.5 + 0.8 (mean ± SEM) ng of LTC4 and LTB4, respectively, per 106 cells; ratios of LTC4 to LTB4 ranged from 16 to 149. Eosinophils stimulated with ionophore (2.5 ,M) or phorbol myristate acetate (1 ,ug per ml) metabolized exogenously added LTC4 to products that coeluted on reverse-phase high-performance liquid chromatography with synthetic S-diastereoisomeric LTC4 sulfoxides and 6-trans-LTB4 diastereoisomers, and this metabolic inactivation was inhibited by Lserine or catalase. Ionophore-activated eosinophils purified from three normal donors also preferentially generated LTC4 (38 ± 3 ngper 106cells) relative to LTB4 (6.0 ± 3.1 ng per 106cells), whereas neutrophils from the same donors released LTB4 (48 ± 21 ng per 106 cells) in a >7-fold excess to LTC4. The predominant production by human eosinophils of LTC4 with its potent smooth muscle spasmogenic and vasoactive properties may contribute to the pathobiology of allergic and other diseases associated with eosinophilia.Human polymorphonuclear leukocytes, activated with diverse stimuli, oxidatively metabolize arachidonic acid by the 5-lipoxygenase-dependent pathway to 5,6-trans-oxido-7,9-trans-11, 14-cis-icosatetraenoic acid (leukotriene A4, LTA4) (1), which in turn is converted enzymatically to (5S,6R)-5,6-dihydroxy-6,14-cis-8, 10-trans-icosatetraenoic acid (leukotriene B4, LTB4) or to (5S,6R)-5-hydroxy-6-S-glutathionyl-7,9-trans-11, 14-cis-icosatetraenoic acid (leukotriene C4, LTC4) (2-4). LTB4 is a potent chemoattractant and aggregating stimulus for both neutrophilic and eosinophilic polymorphonuclear leukocytes (5, 6), and LTC4 is exquisitely active as a spasmogenic and vasoactive substance when administered locally to human airways and skin, respectively (7,8).Human polymorphonuclear leukocytes, predominantly neutrophils, when stimulated with the calcium ionophore A23187 produce LTB4 in marked preference to the sulfidopeptide leukotrienes, LTC4 and its peptide cleavage products (5S,6R)-5-hydroxy-6-S-cysteinylglycyl-7,9-trans -11, 14 -cis-icosatetraenoic acid (leukotriene D4, LTD4) and (5S,6R)-5-hydroxy-6-Scysteinyl-7,9-trans-11,14-cis-icosatetraenoic acid (leukotriene E4, LTE4) (2,4,9,10 diastereoisomers, and the S-diastereoisomeric sulfoxides of LTC4 were prepared as described (11)(12)(13)(14).Cell Purification. Human neutrophils were prepared from citrate-anticoagulated blood of normal volunteer donors by dextran sedimentation of erythrocytes, centrifugation on cushions of Ficoll/Hypaque, and hypotonic lysis of erythrocytes (15). Human eosinophils were obtained from the citrate-anticoagulated b...

Thomas Thorpe's brief greeting to “Mr. W. H.” in the 1609 Quarto of Shakespeares Sonnets has been one of the great conundrums of modern literary studies. But it is not Thorpe's only such greeting to survive. His remaining epistles, taken together with the dedications in many other English books of the period, suggest that, unless Thorpe was here forsaking the conventions that elsewhere governed his and his contemporaries' practice, scholars have been wrong about “the only begetter of these ensuing sonnets,” wrong about “Mr. W. H.,” and wrong about “our ever-living poet” and the “eternity” he “promised.” But in this they are not alone. The original compositor also got something wrong. If the evidence of other Renaissance epistles is to be trusted, the mysterious and celebrated “Mr. W. H.” is a misprint.

Plasma membranes of human neutrophils were solubilized in buffer containing a nonionic detergent and applied to a formylmethionylleucylphenylalanine (fMet-Leu-Phe)-Sepharose column that was washed and eluted with the chemotactic peptide fMet-Leu-Phe. Analysis of the eluate by filtration on Bio-Gel P150 in sodium dodecyl sulfate (NaDodSO4) buffer and by NaDodSO4-polyacrylamide gel electrophoresis revealed three predominant membrane proteins of approximate molecular weight 94 000 (MP-1), 68 000 (MP-2), and 40 000 (MP-3), of which MP-2 accounted for 74--93% of the total protein. Purified MP-1 and MP-2 contained an above average content of hydrophobic amino acids, while MP-2 and MP-3 had an above average content of acid and/or amide amino acids and a below average content of basic amino acids. MP-2 and MP-3, but not MP-1, bound [3H]fMet-Leu-Phe in equilibrium dialysis chambers. Both MP-2 and MP-3 exhibited high-affinity sites with a valence of 0.2--0.3 and mean KA values of 9 x 10(8) and 2 x 10(7) M-1, respectively, and low-affinity sites with a valence of 0.3--0.5 and mean KA values of 3 x 10(7) and 2 x 10(6) M-1 (n = 3). The specificity of the binding of fMet-Leu-Phe was suggested by the failure of MP-2 and MP-3 to bind lipid chemotactic factors and to adhere to a Sepaharose column to which had been coupled chemotactic fragments of the fifth component of complement. A series of synthetic formylmethionyl peptides exhibited the same rank order of potency as inhibitors of the binding of [3H]fMet-Leu-Phe by MP-2 and as stimuli of neutrophil chemotaxis. Membrane proteins isolated by fMet-Leu-Phe-Sepharose affinity chromatography may represent constituents of specific human neutrophil receptors for chemotactic peptides.

To initiate studies on the significance of filarial arachidonic acid metabolism in the immunopathogenesis of human filariasis, we evaluated the ability of microfilariae of the human filarial parasite Brugia malayi to take up and incorporate exogenous arachidonate. When 4 X 10(5) microfilariae were incubated in vitro with 3 nM [3H]arachidonic acid for 0.2, 24, and 48 hr, 23%, 70%, and 75% of tritium activity were associated with microfilariae, respectively. [3H]arachidonic acid was taken up by viable but not by killed microfilariae. Electron microscopic autoradiographic examination of living microfilariae incubated with [3H]arachidonic acid demonstrated numerous tritium-induced silver grains over sectioned parasites. Chromatographic resolution and quantitation of classes of neutral lipids and phospholipids of parasites established that incorporated [3H]arachidonic acid was rapidly and almost completely esterified into these lipids, predominantly into phosphatidylinositol and phosphatidylcholine. Microfilariae, the blood-borne stage of B. malayi, possess the requisite biochemical pathways to rapidly take up and incorporate exogenous arachidonate.

No abstract