Lipopolysaccharide (LPS), an outer-membrane component of Gram-negative bacteria, interacts with LPS-binding protein and CD14, which present LPS to toll-like receptor 4 (refs 1, 2), which activates inflammatory gene expression through nuclear factor kappa B (NF kappa B) and mitogen-activated protein-kinase signalling. Antibacterial defence involves activation of neutrophils that generate reactive oxygen species capable of killing bacteria; therefore host lipid peroxidation occurs, initiated by enzymes such as NADPH oxidase and myeloperoxidase. Oxidized phospholipids are pro-inflammatory agonists promoting chronic inflammation in atherosclerosis; however, recent data suggest that they can inhibit expression of inflammatory adhesion molecules. Here we show that oxidized phospholipids inhibit LPS-induced but not tumour-necrosis factor-alpha-induced or interleukin-1 beta-induced NF kappa B-mediated upregulation of inflammatory genes, by blocking the interaction of LPS with LPS-binding protein and CD14. Moreover, in LPS-injected mice, oxidized phospholipids inhibited inflammation and protected mice from lethal endotoxin shock. Thus, in severe Gram-negative bacterial infection, endogenously formed oxidized phospholipids may function as a negative feedback to blunt innate immune responses. Furthermore, identified chemical structures capable of inhibiting the effects of endotoxins such as LPS could be used for the development of new drugs for treatment of sepsis.

Up-regulation of the CC chemokines and their respective receptors in adipose tissue occurs in human obesity and is associated with increased systemic inflammation.

Polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (20:5 (n-3)) inhibit T lymphocyte activation probably by displacing acylated signaling proteins from membrane lipid rafts. Under physiological conditions, saturated fatty acyl residues of such proteins partition into the cytoplasmic membrane lipid leaflet with high affinity for rafts that are enriched in saturated fatty acyl-containing lipids. However, the biochemical alteration causing displacement of acylated proteins from rafts in PUFA-treated T cells is still under debate but could principally be attributed to altered protein acylation or changes in raft lipid composition. We show that treatment of Jurkat T cells with polyunsaturated eicosapentaenoic acid (20:5 (n-3)) results in marked enrichment of PUFAs (20:5; 22:5) in lipids from isolated rafts. Moreover, PUFAs were significantly incorporated into phosphatidylethanolamine that predominantly resides in the cytoplasmic membrane lipid leaflet. Notably, palmitate-labeled Src family kinase Lck and the linker for activation of T cells (LAT) were both displaced from lipid rafts indicating that acylation by PUFAs is not required for protein displacement from rafts in PUFA-treated T cells. In conclusion, these data provide strong evidence that displacement of acylated proteins from rafts in PUFA-treated T cells is predominantly due to altered raft lipid composition.

Abstract-Membrane vesicles (MVs) released from activated cells and blebs from apoptotic cells are increased in patients with vascular disease and in those with atherosclerotic lesions, and their contribution to inflammatory reactions has been suggested. At sites of inflammation, MVs could serve as rapidly available substrates for peroxidation, carry oxidized compounds to activate other cells, and amplify inflammation. Here, we show that MVs released from tert-butyl hydroperoxide-treated endothelial cells (ECs) and apoptotic blebs, but not MVs from Ca 2ϩ ionophore-treated ECs, stimulate monocyte adhesion to ECs, an important step in atherogenesis. We show that oxidized phospholipids, such as the previously identified 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC), are responsible for biological activity in MVs and apoptotic blebs. Natural antibodies from apolipoprotein E-null mice that recognize POVPC also recognize oxidized MVs, and pretreatment of MVs with these antibodies inhibits their ability to activate ECs. Furthermore, the biological activity of oxidized MVs is inhibited by platelet-activating factor receptor antagonists, which have been shown to inhibit the action of POVPC. Taken together, we show that oxidized MVs and apoptotic blebs stimulate ECs to specifically bind monocytes, with oxidized phospholipids (POVPC) being the active principle. In addition to oxidized lipoproteins, oxidized MVs and apoptotic blebs may play an important role in chronic inflammatory diseases, such as atherosclerosis.

IntroductionTissue factor (TF) is a cell surface receptor initiating blood coagulation, 1 thereby promoting thrombotic events in atherosclerosis, sepsis, and cancer. 2,3 Enhanced endothelial TF expression has been demonstrated in atherosclerotic plaques, 4,5 a process that may account for thrombotic events associated with early and advanced atherosclerosis. TF expression in endothelial cells (ECs) can be induced by a variety of agonists, including inflammatory cytokines, angiogenic growth factors, infectious agents, and minimally modified low-density lipoprotein (MM-LDL). 1,6 MM-LDL regulates TF expression at the level of transcription 5 ; however, the signaling pathways and transcription factors involved in this process are not known. Some of the effects of MM-LDL can be mimicked by oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC). 7 Three biologically active components of oxidized PAPC (Ox-PAPC) have been structurally identified as 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), 8 and 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC). 9 Which of these components of MM-LDL is responsible for induction of TF is not known.In contrast to interleukin-1 (IL-1) or tumor necrosis factor ␣ (TNF-␣), MM-LDL neither up-regulates E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1), nor stimulates neutrophil binding to human ECs. 7 This suggests that classical inflammatory agonists and MM-LDL activate different signaling mechanisms. The major pathway induced by inflammatory cytokines activates transcription factors of the nuclear factor-B (NF-B) family. 10 Whether NF-B is activated by MM-LDL is a subject of controversy. 6,11 In fact, it was shown that MM-LDL and some of its components were capable of down-regulating NF-B-mediated transcription induced by inflammatory cytokines. 12 Thus, the role of the NF-B pathway in inflammatory activation of ECs by oxidized lipids requires further investigation.Apart from NF-B, 13 transcription of the TF gene can be promoted by early growth response factor 1 (EGR-1) and nuclear factor of activated T cells (NFAT). 14,15 Whereas inflammatory cytokines induce NF-B as well as EGR-1, vascular endothelial In the present study, we investigated signaling pathways and transcription factors mediating induction of TF expression in human ECs by biologically active oxidized phospholipids. We show that expression of TF is elevated by OxPAPC, and that this induction was mainly mediated by EGR-1-and NFAT-dependent transcription, but was independent of NF-B activation. Upstream mechanisms activated by OxPAPC were elevation of cytosolic Ca ϩϩ , activation of protein kinase C (PKC), and the mitogenactivated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK MAP kinase cascade. Materials and methods MaterialsCyclosporin A was purchased from Novartis (Vienna, Austria); TNF-␣ from Genzyme (Cambridge...