Phospholipid Ozonation Products Activate the 5-Lipoxygenase Pathway in Macrophages

Abstract

Ozone is a highly reactive environmental toxicant that can react with the double bonds of lipids in pulmonary surfactant. This study was undertaken to investigate the proinflammatory properties of the major lipid-ozone product in pulmonary surfactant, 1-palmitoyl-2-(9'-oxo-nonanoyl)-glycerophosphocholine (16:0/9al-PC), with respect to eicosanoid production. A dose-dependent increase in the formation of 5-lipoxygenase (5-LO) products was observed in murine resident peritoneal macrophages (RPM) and alveolar macrophages (AM) upon treatment with 16:0/9al-PC. In contrast, the production of cyclooxygenase (COX) derived eicosanoids did not change from basal levels in the presence of 16:0/9al-PC. When 16:0/9al-PC and the TLR2 ligand, zymosan, were added to RPM or AM, an enhancement of 5-LO product formation along with a concomitant decrease in COX product formation was observed. Neither intracellular calcium levels nor arachidonic acid release was influenced by the addition of 16:0/9al-PC to RPM. Results from mitogen-activated protein kinase (MAPK) inhibitor studies and direct measurement of phosphorylation of MAPKs revealed that 16:0/9al-PC activates the p38 MAPK pathway in RPM, which results in the activation of 5-LO. Our results indicate that 16:0/9al-PC has a profound effect on the eicosanoid pathway, which may have implications in inflammatory pulmonary disease states where eicosanoids have been shown to play a role.

Figure 1. 16:0/9al-PC activates the 5-LO pathway in resident peritoneal macrophages

Dose response of the production of A) 5-LO products (LTC₄, 6-trans-LTB₄, LTB₄, and 5-HETE) and B) COX products (PGE₂ and TXB₂) with the addition of 16:0/9al-PC (closed squares) and POPC (closed triangles) to RPM for 1h at 37°C. The insets show the amounts of each individual 5-LO metabolite (LTC₄, 6-trans-LTB₄, LTB₄, and 5-HETE) and COX metabolite (PGE₂ and TXB₂) produced with 75 µM 16:0/9al-PC treatment. After incubation, the eicosanoids (LTC₄, 6-trans-LTB₄, LTB₄, 5-HETE, PGE₂, and TXB₂) were analyzed by LC-MS/MS and quantified using standard isotope dilution. Results shown are averages ± SEM (n=3) from three independent experiments. * p< 0.05; ** p<0.01; *** p<0.0001.

Figure 2. 16:0/9al-PC influences eicosanoid production in zymosan treated resident peritoneal macrophages

Dose response of the production of A) 5-LO products (LTC₄, 6-trans-LTB₄, LTB₄, and 5-HETE) and B) COX products (PGE₂ and TXB₂) with the addition of 16:0/9al-PC (closed squares) and POPC (closed triangles) for 3 min followed by the addition of zymosan (25 particles per cell) to resident peritoneal macrophages for 1h at 37°C. After incubation, the eicosanoids (LTC₄, 6-trans-LTB₄, LTB₄, 5-HETE, PGE₂, and TXB₂) were analyzed by LC-MS/MS and quantified using standard isotope dilution. Results shown are averages ± SEM (n=3) from three independent experiments. * p< 0.05; ** p<0.01; *** p<0.0001.

Figure 3. Effect of MAPK inhibitors on the production of 5-LO products in resident peritoneal macrophages

RPM were pre-incubated with the MAPK inhibitors (10 µM), SB202190 (SB; p38 MAPK inhibitor), U0126 (U; MEK/ERK inhibitor), and SP600125 (SP; JNK inhibitor), or vehicle control for 30 min and then stimulated with A) 16:0/9al-PC (37.5 µM) and B) 16:0/9al-PC (37.5 µM) and zymosan (25 particles/cell) for 1h. After incubation, the 5-LO-derived eicosanoids (LTC₄, 6-trans-LTB₄, LTB₄, and 5-HETE) were analyzed by LC-MS/MS and quantified using standard isotope dilution. Results shown are averages ± SEM (n=3) from three independent experiments. * p< 0.05; ** p<0.01; *** p<0.0001.

Figure 4. 16:0/9al-PC induces phosphorylation of p38 in resident peritoneal macrophages

RPM were treated with zymosan (25 particles/cell), 16:0/9al-PC (37.5 µM), or 16:0/9al-PC (37.5 µM) and zymosan (25 particles/cell) for 30 min. A) Cell lysates were collected and analyzed by Western blotting with antibodies to phosphorylated forms of p38 and ERK1/2 and β-actin to normalize for equal loading. The data in this figure are representative of 3 independent experiments. The optical densities measured for the control samples were set as 100% and the results shown for B) phospho-p38 and C) phospho-ERK1/2 are averages ± SEM (n=3) from three independent experiments. * p< 0.05; ** p<0.01; *** p<0.0001.