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(Circulation Research. 2009;104:210.)
© 2009 American Heart Association, Inc.

Cellular Biology

Macrophages Generate Reactive Oxygen Species in Response to Minimally Oxidized Low-Density Lipoprotein

Toll-Like Receptor 4– and Spleen Tyrosine Kinase–Dependent Activation of NADPH Oxidase 2

Yun Soo Bae, Jee Hyun Lee, Soo Ho Choi, Sunah Kim, Felicidad Almazan, Joseph L. Witztum, Yury I. Miller

From the Department of Life Sciences (Y.S.B., J.H.L., S.K.), Ewha Womans University, Seoul, Korea; and Department of Medicine (S.H.C., F.A., J.L.W., Y.I.M.), University of California, San Diego.

Correspondence to Yun Soo Bae, Department of Life Sciences, Ewha Womans University, 11-1 Daehyun-Dong, Seodaemoon-Gu, Seoul 120-750, Korea. E-mail baeys{at}ewha.ac.kr

Oxidative modification of low-density lipoprotein (LDL) plays a causative role in the development of atherosclerosis. In this study, we demonstrate that minimally oxidized LDL (mmLDL) stimulates intracellular reactive oxygen species (ROS) generation in macrophages through NADPH oxidase 2 (gp91phox/Nox2), which, in turn, induces production of RANTES and migration of smooth muscle cells. Peritoneal macrophages from gp91phox/Nox2^–/– mice or J774 macrophages in which Nox2 was knocked down by small interfering RNA failed to generate ROS in response to mmLDL. Because mmLDL-induced cytoskeletal changes were dependent on Toll-like receptor (TLR)4, we analyzed ROS generation in peritoneal macrophages from wild-type, TLR4^–/–, or MyD88^–/– mice and found that mmLDL-mediated ROS was generated in a TLR4-dependent, but MyD88-independent, manner. Furthermore, we found that ROS generation required the recruitment and activation of spleen tyrosine kinase (Syk) and that mmLDL also induced phospholipase PLC1 phosphorylation and protein kinase C membrane translocation. Importantly, the phospholipase C1 phosphorylation was reduced in J774 cells expressing Syk-specific short hairpin RNA. Nox2 modulated mmLDL activation of macrophages by regulating the expression of proinflammatory cytokines interleukin-1β, interleukin-6, and RANTES. We showed that purified RANTES was able to stimulate migration of mouse aortic smooth muscle cells and addition of neutralizing antibody against RANTES abolished the migration of mouse aortic smooth muscle cells stimulated by mmLDL-stimulated macrophages. These results suggest that mmLDL induces generation of ROS through sequential activation of TLR4, Syk, phospholipase C1, protein kinase C, and gp91phox/Nox2 and thereby stimulates expression of proinflammatory cytokines. These data help explain mechanisms by which endogenous ligands, such as mmLDL, can induce TLR4-dependent, proatherogenic activation of macrophages.

Key Words: minimally oxidized LDL • reactive oxygen species • NADPH oxidase 2 • RANTES • atherosclerosis

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