New lipids from human neutrophils: formation of esterified 5-lipoxygenase products in vivo in human infection and effects on generation of neutrophil extracellular traps

Stephen Clark1, Christopher Guy1, Martin Scurr1, Christopher Thomas1, Barbara Coles1, Gareth Roberts2, Matthias Eberl1, Simon Jones1, Nicholas Topley1, Sailesh Kotecha3, Valerie O’Donnell1. 1Dept of Infection, Immunity and Biochemistry, Cardiff University, Cardiff CF14 4XN, United Kingdom, 2Institute of Nephrology, Cardiff University, Cardiff CF14 4XN, United Kingdom, 3Dept of Child Health, Cardiff CF14 4XN, United Kingdom.

5-Lipoxygenase (5-LOX) is well known as a source of free acid eicosanoids including 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotrienes that play key roles in infection and allergic responses. We report a new family of 5-LOX derived lipids in primary human neutrophils comprising 5-HETE attached to phospholipids. Precursor tandem mass spectrometry of lipid extracts from activated neutrophils identified four 5-HETE containing ions that were structurally characterized as three phosphatidylethanolamines (PE) and one phosphatidylcholine (PC) (18:0p/5-HETE-PE, 18:1p/5-HETE-PE, 16:0p/5-HETE-PE and 16:0a/5-HETE-PC).

These novel lipids were formed acutely (within 2 minutes) in vitro in response to fMLP, and levels were enhanced two- to three-fold by priming with LPS, GM-CSF or cytochalasin D (for example, 18:0p/5-HETE-PE was increased to 204 ± 13 % (p < 0.05), 246 ± 15 % (p < 0.05) & 249 ± 15 % (p < 0.01) respecitvely; in comparison to fMLP alone). The total amount of esterified 5-HETE was similar to free 5-HETE (0.37 ± 0.14 ng versus 0.55 ± 0.18 ng/106 cells, respectively). 5-HETE-PC was metabolized within 20 min, but 5-HETE-PEs were stable for at least 3 hours after synthesis. They were not released from the cell and were found in nuclear and extra-nuclear membranes. Their generation required calcium mobilization, phospholipase C, cytosolic phospholipase A2, 5-LOX activating protein and 5-LOX, but not sPLA2, iPLA2, MAP kinase, PKC or MEK (for example, when activating with fMLP levels of 18:0p/5-HETE-PE were inhibited 96 ± 2% by calcium chelation (BAPTA-AM & EGTA), 90 ± 1% (p < 0.001) by U-73112, 95 ± 1 (p < 0.001) by cPLA2α inhibitor, 76 ± 1% (p < 0.05) by MK-886 and 90 ± 11% (p < 0.001) by zileuton, but there was little or no inhibition with OOEPC (51 ± 14%), BEL (-7 ± 29), p38 MAPK inhibitor (2 ± 5 %), GF 109203X (-10 ± 1 %) or PD 98059 (11 ± 12)). Furthermore, they were generated by esterification of free 5-HETE formed acutely on activation, rather than direct oxidation of phospholipid (not shown).

5-HETE-PEs and -PC were detected following incubation of human neutrophils with serum-opsonised Staphylococcus epidermidis (S epi) in vitro (0.07 ± 0.02 ng/106 neutrophils), in a murine model of S epi peritonitis (0.41 ± 0.05 ng/106 neutrophils), and also in lavage from human Gram-positive bacterial peritonitis (0.33 ± 0.25 ng/106 neutrophils).

Formation of neutrophil extracellular traps (NETs; a key anti-microbial response whereby neutrophils release web-like structures of DNA decorated with anti-microbial proteins) in vitro was significantly attenuated by addition of exogenous 5-HETE-PE (release of DNA NETs in response to PMA was reduced from 38 ± 2 to 6 ± 1 ng/106 neutrophils, p < 0.001). Consistent with this, inhibition of 5-LOX (with zileuton or MK-886) enhanced NET formation, indicating a suppressive role for HETE-phospholipids in regulation of host-pathogen interactions (zileuton increased PMA-stimulated release of DNA NETs from 23 ± 2 to 40 ± 3 ng/106 neutrophils (p < 0.01), while MK-886 increased release of DNA NETs from 37 ± 3 to 54 ± 7 ng/106 neutrophils (p < 0.05)).

In summary, acute synthesis of 5-HETE-PEs and -PC by neutrophil 5-LOX occurs in response to bacterial products in vitro and in vivo. The data indicate that formation of phospholipid-esterified 5-HETEs is an agonist-receptor regulated event in granulocytes that may contribute to eicosanoid signalling in infection. A new family of bioactive lipids is structurally characterised, and mechanisms of their formation by human neutrophils in response to bacteria and their biological action is demonstrated.

Data are representative of at least three separate donors, with samples run in triplicate for each experiment. Data are expressed as mean ± SEM. The statistical significance of the difference between sets of data was assessed one-way ANOVA followed by Bonferroni multiple comparisons test. p < 0.05 was considered significant.

The Wellcome Trust, the British Lung Foundation and the EC (FP7) supported this work.