Eoxins are proinflammatory arachidonic acid metabolites produced via the 15-lipoxygenase-1 pathway in human eosinophils and mast cells

Abstract

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)C4 in 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-LTD4 and 14,15-LTE4 in 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 -E4 instead 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 EXC4 after challenge with the proinflammatory agents LTC4, prostaglandin D2, and IL-5, demonstrating that EXC4 can be synthesized from the endogenous pool of arachidonic acid. EXs induced increased permeability of endothelial cell monolayer in 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 LTC4 and 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.

Fig. 1.

Formation of arachidonic acid-derived products in human eosinophils. Isolated eosinophils (10 × 10⁶ cells) were resuspended in 1 ml of PBS and preincubated for 2 min at 37°C before stimulation with 10 μM arachidonic acid for 5 min at 37°C. The reaction was terminated by the addition of 3 vol of methanol. After evaporation, the sample was resuspended in a mobile phase and subjected to analysis in a RP-HPLC/UV system by using AcN:MeOH:H₂O:HAc (29:19:72:0.8 by volume), pH 5.6, as a mobile phase. Arrows indicate the retention times of synthetic standards. (Inset) UV spectrum of metabolite 1.

Fig. 2.

LC-MS/MS analysis of cysteinyl-containing 15-LO-derived products. Shown is LC-MS/MS spectrum of metabolite 1 formed by eosinophils after incubation with arachidonic acid compared with spectra of synthetic 14,15-LTC₄ (EXC₄) and LTC₄ standards. (Top) The spectrum of the product ion scan of 626.2 [M+H]⁺ derived from a metabolite 1 formed in eosinophils after challenge with arachidonic acid (10 μM) for 5 min at 37°C. (Middle) The spectrum of synthetic 14,15-LTC₄ (EXC₄) standard. (Bottom) The spectrum of synthetic LTC₄ standard.

Fig. 3.

Fragments distinguishing of cysteinyl 14,15-LTs from cysteinyl LTs in product ion spectra. A prominent difference in the positive-ion mode MS/MS product ion spectra of 5-LO-derived cysteinyl LTs and cysteinyl 14,15-LTs (EXs) are m/z 189 and 205. The probable origins of these fragment ions are indicated.

Fig. 4.

Alterations in vascular permeability induced by EXs. (A) Analysis of rapid changes in EC barrier function in response to EXC₄, EXD₄, EXE₄, LTC₄, LTB₄, and histamine. Analyses were accomplished by continuous registration of TEER with electrodes on each side of the EC monolayer. The maximal decrease in resistance in this model varies typically at approximately −70%. (B) Time course for changes in EC barrier function induced by EXD₄ (10⁻⁸ and 10⁻⁷ M, respectively) in comparison with LTD₄ (10⁻⁸ M). Each value represents the mean value of triplicate determinations ± SD.

Fig. 5.

Proposed metabolic pathway for the formation of EXs.