Lipoxins are trihydroxytetraene metabolites derived through a double lipoxygenation of arachidonic acid. Lipoxin A4 (LXA4) was prepared by total chemical synthesis, and its capacity to modulate eosinophil migration has been evaluated. LXA4 is a weak and partial chemotactic agent; at 10(-6) M, it achieved about 20% of the response of 10(-6) M platelet-activating factor (PAF). Preincubation of eosinophils with increasing doses of LXA4 (10(-10)-10(-5) M) resulted in a concentration-dependent inhibition of cell migration induced by 10(-6) M formyl-methionyl-leucyl-phenylalanine (FMLP) and 10(-6) M PAF. The concentration of LXA4 which produced 50% inhibition (IC50) of eosinophil migration was approximately 10(-6) M. LXA4 (10(-10)-10(-6) M) did not elicit ECP release or modulate ECP release induced by 10(-6) M FMLP. LXA4 may have antiallergic properties in preventing eosinophilic migration.

1 To test the hypothesis that protein kinase C (PKC) is involved in the inhibitory actions of lipoxin A4 (LXA4) on second messenger generation, we studied the effects of LXA4 on PKC in human neutrophils and on leukotriene B4 (LTB4)-stimulated inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) generation. 2 LXA4, 1 uM, caused a fall in cytosolic PKC-dependent histone phosphorylating activity to 23.5% of basal levels. 3 LXA4, caused an increase in particulate PKC-dependent histone phosphorylating activity with a bellshaped dose-response fashion; maximal stimulation was observed at 10 nM LXA4. 4 Western blot analysis with affinity-purified antibodies to a-and f-PKC showed that only the f-PKC isotype was translocated by LXA4.5 LXA4 inhibited LTB4-stimulated Ins(1,4,5)P3 generation in a bell-shaped fashion with maximal inhibition at 1 nM LXA4. The observed inhibition was dose-dependently removed by pre-incubation with a PKC inhibitor (Ro-31-8220). 6 These results show that LXA4 activates PKC in whole cells and supports a role for PKC activation in the inhibitory action of LXA4 on LTB4-induced Ins(1,4,5)P3 generation.7 LXA4 (1-1000 nM) pre-incubation did not affect specific binding of [3H]-LTB4 to neutrophils. Thus, the inhibitory effect of LXA4 on LTB4-stimulated Ins(1,4,5)P3 generation could not be attributed to an effect on LTB4 receptors.

The biological activities of chemically synthesized leukotriene B, and eight structural analogues have been studied using chemotaxis, lysosomal-enzyme release and receptor-binding assays on human neutrophils. The results show that increasing the number of double bonds between C14 and C20, having triple bonds at C6 or C14, substitution of the primary carboxyl group at C1, changing the geometry of the double bond at C6 from the cis to trans configuration and changing the chirality of the hydroxyl group at C12 from the R to the S configuration result in substantial loss of both biological activity and the capacity to bind to the LTB, recognition site in parallel. We suggest that the functional epitopes of 5S, 12R-dihydroxy-6,14-cis-8,lO-truns-icosatetraenoic acid (LTB,) are either the same, or reside in the same domain as the binding site for the LTB, receptor. Development of LTB, antagonists to the high-affinity LTB, receptor, based on the structure of LTB,, is unlikely to be successful.Arachidonic acid is released from membrane phospholipids upon stimulation and is transformed via 5-hydroperoxy-6,8,11,14-icosatetraenoic acid (5-HPETE) by the 5-lipoxygenase enzyme [l-31 into an unstable epoxide, leukotriene A, (LTA,; 5,6-oxido-7,9,11,14-icosatetraenoic acid) [4]. LTA, is formed from 5-HPETE by abstraction of a hydrogen at ClO and elimination of a hydroxyl anion from the hydroperoxy group. LTA, undergoes either spontaneous, non-enzymic hydrolysis to form several stereochemically distinct 5,12-dihydroxy-icosatetraenoic acids (5,12-diHETE) or is enzymically catalyzed by the hydrolase enzyme to form leukotriene B, (LTB, ; 5S, 12R-dihydroxy-6,14-cis-8,1 O-trunsicosatetraenoic acid), a specific form of 5,12-diHETE [5, 61. Conjugation of LTA, with glutathione at the C6 position, by the glutathione-S-transferase enzyme LTC, synthetase, leads to the formation of LTC, (5S-hydroxy-GR-glutathionyl-7,9-truns-ll,14-cis-icosatetraenoic acid) [7, 81. LTC, is metabolically altered by successive elimination of a y-glutamyl residue, then glycine, to form its 6-cysteinylglycyl analogue, LTD, [9] and its 6-cysteinyl analogue, LTE, [lo] respectively. The structure of LTB, is 5S,12R-dihydroxy-6,14-cis-8,1O-truns-icosatetraenoic acid (compound I) [ll]. It is synthesized by human neutrophils, monocytes and alveolar macrophages in response to a wide variety of stimuli Abbreviations. PMN, polymorphonuclear cells ; HPETE, hydroperoxyicosatetraenoic acid; LTA,, 5,6-oxido-7,9,11,14-icosatetraenoic acid; diHETE, dihydroxyicosatetraenoic acids ; DMA, dimethylamide; LTB,, 5s. 12R-dihydroxy-6,14-cis-8,lO-trunL~-icosatetraenoic acid (compound 1); LTC,, SS-hydroxy-6R-glutathionyl-7,9-trans-11,14-cis-icosatetraenoic acid; IC,,, concentration at which half of the response is inhibited; fmet, formylmethionyl; HBSS, Hanks' balanced-salt solution; HPF, High-power field; compounds I-IX, the structures and chemical formulae are shown in Fig. 1. and is a potent chemotactic agent for neutrophils [12]. It induces aggregation of polymorphonuclear cells (P...