Recently, the cloning of a novel Ca²⁺-independent phospholipase A₂(iPLA₂) from Chinese hamster ovary cells as well as from mouse and rat sources containing a C-terminal lipase motif and eight N-terminal ankyrin repeats has been described. In this report we describe the cloning of the human iPLA₂ cDNA and its expression in B-cells and show that the iPLA₂ gene undergoes extensive alternative splicing generating multiple isoforms that contribute to a novel mechanism to control iPLA₂activity. The full-length cDNA clone encodes a 806-amino acid protein with a calculated molecular mass of 88 kDa. The protein contains a lipase motif, GXSXG, and ankyrin repeats, as described for the hamster and rodent forms of the enzyme but has an additional 54-amino acid proline-rich insertion in the last of the eight ankyrin repeats (residues 395–449). Furthermore, at least three additional isoforms most likely due to alternative splicing were identified. One that is present as a partial cDNA in the expressed sequence tag data base is similar to iPLA₂ but terminates just after the lipase active site, and two other isoforms contain only the iPLA₂ ankyrin repeat sequence (ankyrin-iPLA₂-1 and -2). Ankyrin repeats are involved in protein-protein interactions and because the purified iPLA₂enzyme exists as a multimeric complex of 270–350 kDa, the expression of just the ankyrin-iPLA₂ sequence suggested that these may also interact with the iPLA₂ oligomeric complexes and perhaps modulate PLA₂ activity. Transfection of the human iPLA₂ cDNA into COS cells resulted in a substantial increase in calcium-independent PLA₂ activity in cell lysate. No activity above background was observed following ankyrin-iPLA₂-1 cDNA transfection. However, co-transfection of the ankyrin-iPLA₂-1 and the iPLA₂ cDNAs resulted in a 2-fold reduction in activity compared with iPLA₂ alone. A similar co-transfection of ankyrin-iPLA₂-1 cDNA with the cPLA₂cDNA had no effect on PLA₂ activity. These results suggest that the ankyrin-iPLA₂ sequence can function as a negative regulator of iPLA₂ activity and that the alternative splicing of the iPLA₂ gene can have a direct effect on the attenuation of enzyme activity.

Human eosinophils contain abundant amounts of 15-lipoxygenase (LO)-1. The biological role of 15-LO-1 in humans, however, is unclear. Incubation of eosinophils with arachidonic acid led to formation of a product with a UV absorbance maximum at 282 nm and shorter retention time than leukotriene (LT)C₄in reverse-phase HPLC. Analysis with positive-ion electrospray tandem MS identified this eosinophil metabolite as 14,15-LTC₄. This metabolite could be metabolized to 14,15-LTD₄and 14,15-LTE₄in eosinophils. Because eosinophils are such an abundant source of these metabolites and to avoid confusion with 5-LO-derived LTs, we suggest the names eoxin (EX)C₄, -D₄, and -E₄instead of 14,15-LTC₄, -D₄, and -E₄, respectively. Cord blood-derived mast cells and surgically removed nasal polyps from allergic subjects also produced EXC₄. Incubation of eosinophils with arachidonic acid favored the production of EXC₄, whereas challenge with calcium ionophore led to exclusive formation of LTC₄. Eosinophils produced EXC₄after challenge with the proinflammatory agents LTC₄, prostaglandin D₂, and IL-5, demonstrating that EXC₄can be synthesized from the endogenous pool of arachidonic acid. EXs induced increased permeability of endothelial cell monolayerin vitro, indicating that EXs can modulate and enhance vascular permeability, a hallmark of inflammation. In this model system, EXs were 100 times more potent than histamine and almost as potent as LTC₄and LTD₄. Taken together, this article describes the formation of proinflammatory EXs, in particular in human eosinophils but also in human mast cells and nasal polyps.

Incubation of human endothelial cells with leukotriene A₄ resulted in the formation of leukotrienes B₄, C₄, D₄ and E₄. Endothelial cells did not produce leukotrienes after stimulation with the ionophore A23187 and/or exogenously added arachidonic acid. However, incubation of polymorphonuclear leukocytes with ionophore A23187 together with endothelial cells led to an increased synthesis of cysteinyl‐containing leukotrienes (364%, mean, n= 11) and leukotriene B₄ (52%) as compared to leukocytes alone. Thus, the major part of leukotriene C₄ recovered in mixed cultures was attributable to the presence of endothelial cells. Similar incubations of leukocytes with fibroblasts or smooth muscle cells did not cause an increased formation of leukotriene C₄ or leukotriene B₄.The increased biosynthesis of cysteinyl‐containing leukotrienes and leukotriene B₄ in coincubations of leukocytes and endothelial cells appeared to be caused by two independent mechanisms. First, cell interactions resulted in an increased production of the total amount of leukotrienes, suggesting a stimulation of the leukocyte 5‐lipoxygenase pathway, induced by a factor contributed by endothelial cells.Secondly, when endothelial cells prelabeled with [³⁵S]cysteine were incubated with either polymorphonuclear leukocytes and A23187, or synthetic leukotriene A₄, the specific activity of the isolated cysteinyl‐containing leukotrienes were similar. Thus, transfer of leukotriene A₄ from stimulated leukocytes to endothelial cells appeared to be an important mechanism causing an increased formation of cysteinyl‐containing leukotrienes in mixed cultures of leukocytes and endothelial cells.In conclusion, the present study indicates that the vascular endothelium, when interacting with activated leukocytes, modulates both the quantity and profile of liberated leukotrienes.