Lipid bodies in oxidized LDL-induced foam cells are leukotriene-synthesizing organelles: a MCP-1/CCL2 regulated phenomenon Adriana R. Silva a , Patricia Pacheco b , Adriana Vieira-de-Abreu b , Clarissa M. Maya-Monteiro b , Barbara D'Alegria a,b , Kelly G. Magalhães b , Edson F. de Assis b , Christianne Bandeira-Melo c , Hugo C. Castro-Faria-Neto b , Patricia T. Bozza b, ⁎ a Laboratório de Inflamação, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil b Laboratorio de Imunofarmacologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz; Av. Brasil 4365, Manguinhos, 21045-900 Rio de Janeiro, RJ, Brazil c Laboratório de Inflamação, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil abstract article info Article history: Received 23 September 2008 Received in revised form 9 June 2009 Accepted 23 June 2009 Available online 30 June 2009 Keywords: Atherosclerosis Foam cell Lipid droplet Lipoxygenase Leukotriene Inflammation MCP-1/CCL2 ERK Macrophage Lipid-laden foam macrophages are emerging as key players in early atherogenesis. Even though cytoplasmic lipid bodies (lipid droplets) are now recognized as organelles with cell functions beyond lipid storage, the mechanisms controlling lipid body biogenesis within macrophages and their additional functions in atherosclerosis are not completely elucidated. Here we studied oxLDL-elicited macrophage machinery involved in lipid body biogenesis as well as lipid body roles in leukotriene (LT) synthesis. Both in vivo and in vitro, oxLDL (but not native LDL) induced rapid assembly of cytoplasmic lipid bodies-bearing ADRP within mice macrophages. Such oxLDL-elicited foamy-like phenotype was a pertussis toxin-sensitive process that depended on a paracrine activity of endogenous MCP-1/CCL2 and activation of ERK. Pretreatment with neutralizing anti-MCP-1/CCL2 inhibited macrophage ADRP protein expression induced by oxLDL. By directly immuno-localizing leukotrienes at their sites of synthesis, we showed that oxLDL-induced newly formed lipid bodies function as active sites of LTB 4 and LTC 4 synthesis, since oxLDL-induced lipid bodies within foam macrophages compartmentalized the enzyme 5-lipoxygenase and five lipoxygenase-activating protein (FLAP) as well as newly formed LTB 4 and LTC 4 . Consistent with MCP-1/CCL-2 role in ox-LDL- induced lipid body biogenesis, in CCR2 deficient mice both ox-LDL-induced lipid body assembly and LT release were reduced as compared to wild type mice. In conclusion, oxLDL-driven foam cells are enriched with leukotriene-synthesizing lipid bodies – specialized organelles whose biogenic process is mediated by MCP-1/CCL2-triggered CCR2 activation and ERK-dependent downstream signaling – that may amplify inflammatory mediator production in atherosclerosis. © 2009 Elsevier B.V. All rights reserved. 1. Introduction A hallmark of atherosclerosis is the fatty streak, an accumulation of lipid-laden macrophages namely foam cells beneath the endothelial layer in arteries. Since it is the earliest recognizable lesion of atherosclerosis, much attention is focused on understanding the etiology of this phenomenon and the roles of foam cells in disease. Uncontrolled oxLDL uptake by macrophages through scavenger receptors causes triglyceride and cholesterol loading, followed by cholesterol esterification and storage of cholesteryl esters (CEs) in cytoplasmic lipid bodies or lipid droplets [1–4]. Cytoplasmic lipid bodies are increasingly recognized as dynamic and functionally active organelles [5–7]. Lipid bodies in macrophages are composed of triglycerides and cholesterol esther enriched core, which is surrounded by a monolayer of phospholipids. In addition to lipids, a variable and diverse group of proteins have been found associated to lipid bodies. Perilipin, adipose differentiation related protein (ADRP) and TIP47 – members of the PAT family – are – structural proteins considered essential for lipid storage and metabo- lism (reviewed in [8]). ADRP is associated with cytoplasmic lipid bodies in all types of cells examined and is described as a specific protein marker for lipid bodies [9,10], including in monocyte/ macrophages [11–13]. In addition, ADRP has been implicated in atherosclerosis progression as increased ADRP levels are observed in atherogenic plaques [14,15] and ADRP gene deletion leads to reduced Biochimica et Biophysica Acta 1791 (2009) 1066–1075 Abbreviations: ADRP, adipose differentiation related protein; AA, arachidonic acid; CCR2, CC chemokine receptor 2; COXs, cyclooxygenases; cPLA 2 , cytosolic phospholipase A 2 ; cysLTs, cysteinyl leukotrienes; ERK, extracellular signal-regulated kinase; FLAP, five lipoxygenase-activating protein; oxLDL, oxidized low density lipoprotein; LO, lipoxy- genase; LTs, leukotrienes; LPS, lipopolysaccharide; IL, interleukin; i.p., intraperitoneal; MCP-1/CCL2, monocyte chemotactic protein-1/CC Ligand 2; MAP, mitogen-activated protein; PAF, platelet-activating factor; WT, wild type ⁎ Corresponding author. E-mail address: pbozza@ioc.fiocruz.br (P.T. Bozza). 1388-1981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2009.06.004 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbalip