Abstract
Secreted phospholipase A2 group X (sPLA(2)-X) is one of the most potent enzymes of the phospholipase A(2) lipolytic enzyme superfamily. Its high catalytic activity toward phosphatidylcholine (PC), the major phospholipid of cell membranes and low-density lipoproteins (LDL), has implicated sPLA(2)-X in chronic inflammatory conditions such as atherogenesis. We studied the role of sPLA(2)-X enzyme activity in vitro and in vivo, by generating sPLA(2)-X-overexpressing macrophages and transgenic macrophage-specific sPLA(2)-X mice. Our results show that sPLA(2)-X expression inhibits macrophage activation and inflammatory responses upon stimulation, characterized by reduced cell adhesion and nitric oxide production, a decrease in tumor necrosis factor (TNF), and an increase in interleukin (IL)-10. These effects were mediated by an increase in IL-6, and enhanced production of prostaglandin E(2) (PGE(2)) and 15-deoxy-Delta12,14-prostaglandin J(2) (PGJ(2)). Moreover, we found that overexpression of active sPLA(2)-X in macrophages strongly increases foam cell formation upon incubation with native LDL but also oxidized LDL (oxLDL), which is mediated by enhanced expression of scavenger receptor CD36. Transgenic sPLA(2)-X mice died neonatally because of severe lung pathology characterized by interstitial pneumonia with massive granulocyte and surfactant-laden macrophage infiltration. We conclude that overexpression of the active sPLA(2)-X enzyme results in enhanced foam cell formation but reduced activation and inflammatory responses in macrophages in vitro. Interestingly, enhanced sPLA(2)-X activity in macrophages in vivo leads to fatal pulmonary defects, suggesting a crucial role for sPLA(2)-X in inflammatory lung disease.
Highlights
Phospholipases have been implicated in the pathogenesis of lipid-mediated inflammatory diseases such as atherosclerosis
In vivo experiments show that enhanced Secreted phospholipase A2 group X (sPLA2-X) activity in macrophages leads to neonatal death due to severe lung pathology characterized by an interstitial pneumonia with infiltration of surfactant-laden alveolar macrophages and edema
Because the Secretory phospholipases A2 (sPLA2)-X protein has previously been detected in macrophage-derived foam cells [9] and spleen macrophages [19] by immunohistochemistry, we investigated functional overexpression of the active sPLA2-X enzyme in macrophages both in vitro and in vivo
Summary
Generation of a Macrophage-specific sPLA2-X Construct—A macrophage-specific sPLA2-X construct was generated by cloning the active human sPLA2-X cDNA (a generous gift from Dr Lambeau) starting at the second methionine initiator site (Met-32 construct) [7] behind the macrophage-specific CD68 promoter (2.9-kb CD68 ϩ IVS-1 sequence) [11] into the pcDNA 3 vector (Invitrogen) using convenient restriction sites (Fig. 1A). Northern Blotting—To verify expression of sPLA2-X in transfected RAW264.7 cells, RNA was isolated from positive and negative sPLA2-X clones using Tri-Reagent (Sigma) according to the manufacturer’s instructions. Phospholipase Activity—Phospholipase activity was measured in culture medium from sPLA2-X and control RAW264.7 cells using a sPLA2 assay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions. Quantitative PCR—For quantitative PCR analysis, total RNA was isolated from hu-sPLA2-X and control cells using Tri-Reagent (Sigma). SPLA2-X and control RAW264.7 cells were plated at a density of 2 ϫ 106 cells in 6-well plates in serum-free medium (Optimem, Invitrogen) constituted with 100 units/ml penicillin, 100 g/ml streptomycin, 2 mM L-glutamine. To determine the presence of human sPLA2-X, lung sections were stained with polyclonal rabbit anti-human sPLA2-X antisera (1:50), a generous gift from Dr Gelb (University of Washington).
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