Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal antiinflammatory drugs and transcellular processing

Abstract

Aspirin therapy inhibits prostaglandin biosynthesis without directly acting on lipoxygenases, yet via acetylation of cyclooxygenase 2 (COX-2) it leads to bioactive lipoxins (LXs) epimeric at carbon 15 (15-epi-LX, also termed aspirin-triggered LX [ATL]). Here, we report that inflammatory exudates from mice treated with omega-3 polyunsaturated fatty acid and aspirin (ASA) generate a novel array of bioactive lipid signals. Human endothelial cells with upregulated COX-2 treated with ASA converted C20:5 omega-3 to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. Each was used by polymorphonuclear leukocytes to generate separate classes of novel trihydroxy-containing mediators, including 5-series 15R-LX(5) and 5,12,18R-triHEPE. These new compounds proved to be potent inhibitors of human polymorphonuclear leukocyte transendothelial migration and infiltration in vivo (ATL analogue > 5,12,18R-triHEPE > 18R-HEPE). Acetaminophen and indomethacin also permitted 18R-HEPE and 15R-HEPE generation with recombinant COX-2 as well as omega-5 and omega-9 oxygenations of other fatty acids that act on hematologic cells. These findings establish new transcellular routes for producing arrays of bioactive lipid mediators via COX-2-nonsteroidal antiinflammatory drug-dependent oxygenations and cell-cell interactions that impact microinflammation. The generation of these and related compounds provides a novel mechanism(s) for the therapeutic benefits of omega-3 dietary supplementation, which may be important in inflammation, neoplasia, and vascular diseases.

Figure 1

Inflammatory exudates from murine dorsal pouches treated with aspirin generate novel compounds: LC/MS/MS. TNF-α–induced leukocyte exudates collected at 6 h from FVB mice given ASA (3.5 h at 500 μg/air pouch) and EPA (4 h at 300 μg/pouch) contained (2.3 ± 0.5 × 10⁶ leukocytes per pouch); see Materials and Methods. (A) Selected ion chromatogram of mono-HEPEs. (B) MS/MS of 18R-HEPE. (C) MS/MS of 5S-HEPE. (D) MS/MS of 12,15,18R-triHEPE (see text for identification of diagnostic ions). Results are representative of n = 4.

Figure 2

Human ECs and PMNs: novel trihydroxy-containing compounds LC/MS/MS. (A) HUVECs treated with IL-1β (24 h) and ASA. (B) Human PMNs (∼ 30 × 10⁶/ml) incubated (30 min, 37°C) with serum-treated zymosan (100 ng/ml) and acetylated ASA-recombinant COX-2–derived products from EPA. (C) MS/MS of 5,12,18R-triHEPE (see text for ions and fragmentation). (D) MS/MS of 15-epi-LXA₅. Results are representative of n = 3–5 for PMNs and n = 3 for ECs.

Figure 3

The novel compounds inhibit human PMN transmigration and inflammation in the murine air pouch. (A) Inhibition of PMN transendothelial migration. PMNs were incubated with isolated compounds [10⁻¹⁰–10⁻⁶ M] 18R-HEPE (○), 5,12,18R-triHEPE (•), or ATL analogue used for reference (♦), and transmigration was initiated using an optimal amount of LTB₄ [10 nM] (100%) and HUVEC coincubations (90 min, 37°C). Results are the mean ± SEM; n = 3. (B) Competition binding with human recombinant LTB₄ receptor. LTB₄ receptor stably expressed in HEK293 cells (∼5 × 10⁵ cells/incubation) were incubated with [³H]LTB₄ (∼1 nM) plus LTB₄ (▪) and 18R-HEPE (○), 5,12,18R-triHEPE (•), or LTB₅ (□). Results are the mean ± SEM from n = 3. (C) Inhibition of leukocyte trafficking in 6 d dorsal air pouch. TNF-α (100 ng) was injected intrapouch and 100 ng i.v. tail vein of test compound. ATLa (analogue 15(S)-16(parafluoro)-phenoxy-LXA₄; reference 16) was used for comparison. Results represent n = 4.

Figure 4

Proposed scheme for generating functional arrays of lipid signals from ω-3 PUFA via transcellular processing: endogenous inhibitors of microinflammation. At sites where COX-2 is upregulated and treated with NSAIDs, PG formation from C20:4 is blocked. Systemic ω-3 PUFAs are converted via a COX-2–NSAID lipoxygenase–type mechanism that abstracts hydrogen in a stereospecific fashion at C16 or C13 in C20:5 to give R insertions of molecular O₂ to yield 15R-H(p)EPE or 18R-H(p)EPE to form epoxides or are reduced to alcohols. The complete stereochemistry for each of the trihydroxy compounds remains to be determined, and the compounds are depicted in their likely configurations. These compounds interact with cells in the local microenvironment, inhibiting PMN recruitment. COX-2–NSAID-dependent hydrogen abstraction and insertion of molecular oxygen occurs with all ω-3 PUFAs containing 1,4-cis pentadiene units tested (see text for details). Thus, sequential oxygenations of ω-3 PUFA during cell–cell interactions generate novel arrays of signals of interest in microinflammation.