Horse eosinophils purified to greater than 98% generated slow reacting substance (SRS) when incubated with the calcium ionophore A23187. On a per cell basis, eosinophils generated four to five times the SRS produced by similarly treated horse neutrophils. Eosinophil SRS production was inhibited by 5,8,11,14-eicosatetraynoic acid and augmented by indomethacin and arachidonic acid, suggesting that it was a product(s) of the lipoxygenase pathway of arachidonic acid metabolism. Compounds with SRS activity were purified by high-pressure liquid chromatography (HPLC) and identified by ultraviolet spectra, spectral shift on treatment with lipoxygenase, incorporation of [14C]arachidonic acid, gas chromatography-mass spectrometry, and comparison of retention times on HPLC to authentic standards. The eosinophil products characterized were 5-(S), 12-(R)-dihydroxy-6-cis-8, 10-trans-14-cis-eicosatetraenoic acid (leukotriene B4) and its 5-(S), 12-(R)-6-trans and 5-(S), 12-(S)-6-trans isomers, 5-(S)-hydroxy-6-(R)-S-glutathionyl-7,9-trans-11, 14-cis-eicosatetraenoic acid (leukotriene C4) and its 11-trans isomer, and 5-(S)-hydroxy-6-(R)-S-cysteinylglycine-7,9-trans-11,14-cis-eicosatetraenoic acid (leukotriene D4).

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To investigate a possible role of phospholipase A, (PLA,) in the respiratory burst in bovine eosinophilic and neutrophilic leukocytes dependent on GTP-binding protein (G-protein), we permeabilized these cells with Staphylococcus aureus a-toxin and induced NADPH oxidase activity with the non-hydrolysable GTP analogue GTP [S] or the aluminium tetrafluoro complex AlF,-. Under same experimental conditions, cells responded with different onset times. The onset time for eosinophils was 50-200 s, for neutrophils it was only a few seconds. GTP[S] stimulated in neutrophils only 5 % of the respiratory burst compared to eosinophils, whereas AlF,--induced comparable responses (neutrophils 120% of eosinophils). GDP inhibited these responses with an IC,, value of 2.4 mM. Arachidonic acid showed, with the exception of AIF,-stimulated neutrophils, on both stimuli and cell types an enhancing effect (150%) that reached its maximum at 0.1-1 pM. The PLA, inhibitor 4-bromophenacylbromide reduced the GTP[S]-and AlF,--induced response almost completely (10 pM) and the inhibition was not significantly different for eosinophils and neutrophils (IC50 1 -3 pM). If the respiratory burst was reduced with 4-bromophenacylbromide to 1 -4% of the original value, 10% of the basal NADPH oxidase activity could be restored by addition of only 20-100 nM arachidonic acid. In addition, the PLA, activator adriamycin enhanced the response in a dose-dependent manner and in the same order as arachidonic acid did. The results presented above suggest that the respiratory burst may be regulated by different low-molecular-mass and/or heterotrimeric G-proteins and an active role for arachidonic acid or its metabolites in the activation and the maintenance of the direct G-protein-stimulated respiratory burst in bovine eosinophils and neutrophils.When stimulated by particulate or soluble stimuli like opsonized zymosan, formyl-methionyl-leucyl-phenylalanine, leuktriene B,, platelet-activating factor or the anaphylatoxine C,a, phagocytes of the immune system generate superoxide (O;-) and other reduced oxygen species by using an NADPH oxidase, for reviews, see [l -31. of Fribourg, me du mussCe 5 , CH-1700 Fribourg, Switzerland cytosolic factors p47ph0" and p67ph"", the small G-protein p21r"'1'2 and an associated GDP dissociation inhibitor that have been identified as components of the activating mechanism at least in the cell-free system [4-61.The phosphorylation of several proteins has been reported. The phosphorylation of the p47phoX occurs in normal cells but has been reported to be absent in patients with autosomal recessive chronic granulomatous disease 171. There have been reports suggesting that phospholipase A, (PLA,) is involved in the activation of the respiratory burst oxidase. An increase in PLA, activity and a release of arachidonic acid has been shown to occur following the stimulation of NADPH oxidase by different stimuli in neutrophils as well as in macrophages [8-101. Quinacrine and 4-bromophenacylbromide, inhibitors of PLA,, have previously be...