#52 Oxidized phosphatidylcholine induces COX2 gene expression and cytokine secretion by human airway smooth muscle cells
Christopher Daniel Pascoe, CHRIM; Mirna MK Ragheb, CHRIM; Gerald L Stelmack, CHRIM; Aruni Jha, CHRIM; Andrew J Halayko, CHRIM
Mechanisms to maintain redox balance in the lungs are overwhelmed in severe asthma, resulting in oxidative stress that damages the phosphatidylcholine (PC)-rich lining of the lung forming oxidized 2-arachidonoyl-1-palmitoyl-sn-glycero-3-phosphocholine (OxPAPC). OxPAPCs are bioactive, with pro-inflammatory effects in numerous chronic diseases. In asthma, though airway smooth muscle (ASM) can orchestrate chronic inflammation, it is not known whether OxPAPCs promote a pro-inflammatory ASM phenotype. Hypothesis: Oxidized phosphatidylcholine induce biogenesis of pro-inflammatory mediators through receptor mediated pathways in ASM cells.
Confluent cultures of primary human ASM cells from ex-smoker donors, were maintained in serum-free medium and treated (24hrs) with OxPAPC (0-160g/mL) or PSPC, a fully saturated, non-oxidizable negative control. Lactate dehydrogenase (LDH) release was assayed to evaluate cytotoxicity and total mRNA and protein collected for qPCR and Western immunoblotting. Cytokine and oxylipin release in cell culture medium was also measured. Values are mean ± SEM. All experiments performed in triplicate.
OxPAPC caused a concentration-dependent increase in LDH release. Release with exposure to 160g/mL of OxPAPC was 13.5 ± 5.4% of maximum positive control (p<0.05 vs. negative control). OxPAPC incubation also dose-dependently increased the abundance of mRNA for COX2. Maximum induction of COX2 mRNA was 6.3 ± 0.78 log2 fold (p<0.05 vs. negative control) with OxPAPC (160g/mL). OxPAPC exposure for 24 hrs also induced significant release of IL-6 (237.6 ± 48.1 pg/mL), IL-8 (58.5 ± 45.7 pg/mL), and GM-CSF (40.1 ± 22.7 pg/mL) and 33 oxylipins (p<0.05 vs. No Treatment). Western blotting data suggests that these effects are not through canonical TLR4 signaling mechanisms.
OxPAPC induces a pro-inflammatory phenotype in human ASM cells that is characterized by accumulation of mRNA for the prostaglandin synthesizing enzyme, COX2, concomitant with induction of cytokines, chemokines, and oxylipins that promote airway neutrophilia. Our data suggest that OxPAPC may underpin chronic airway inflammation in asthma.