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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:1213.)
© 2005 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Toll-Like Receptor 4–Dependent and –Independent Cytokine Secretion Induced by Minimally Oxidized Low-Density Lipoprotein in Macrophages

Yury I. Miller; Suganya Viriyakosol; Dorothy S. Worrall; Agnès Boullier; Susan Butler; Joseph L. Witztum

From the Division of Endocrinology and Metabolism (Y.I.M., D.S.W., A.B., S.B., J.L.W.), Department of Medicine, and Veterans Administration (S.V.), San Diego Healthcare System and Department of Pathology and Medicine, University of California, San Diego.

Correspondence to Yury I. Miller, MD, PhD, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0682. E-mail yumiller{at}ucsd.edu

Objective— Innate immune responses to oxidized low-density lipoprotein LDL (LDL) regulate the development of atherosclerosis. We demonstrated previously that an early form of oxidized LDL, minimally modified LDL (mmLDL), triggers cytoskeletal rearrangements in macrophages via CD14 and Toll-like receptor 4 (TLR4)/MD-2. Because lipopolysaccharide (LPS) activation of TLR4 leads to proinflammatory gene expression, in this study, we asked whether mmLDL also induced proinflammatory signaling.

Methods and Results— We studied cytokine secretion and signaling in J774 and primary peritoneal macrophages stimulated with mmLDL, which was prepared by incubating LDL with cells expressing human 15-lipoxygenase. MmLDL stimulated robust phosphoinositide 3-kinase (PI3K) activation, and Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, which exceeded that induced by LPS. On the other hand, although mmLDL induced nuclear factor B (NF-B) p65 translocation to the nucleus, there was no detectable NF-B activation. However, mmLDL induced early mRNA and protein expression of the cytokines MIP-2, MCP-1, tumor necrosis factor-, and interleukin-6. Chemokine MIP-2 but not MCP-1 secretion depended on TLR4/MyD88, ERK1/2, and PI3K signaling. In turn, TLR4 regulated phosphorylation of ERK1/2 but not of Akt, suggesting that mmLDL-induced PI3K activation is TLR4 independent.

Conclusions— In macrophages, mmLDL activates TLR4-dependent and -independent signaling pathways, resulting in secretion of proinflammatory cytokines. These results provide new insights into the inflammatory origins of atherosclerosis.

Minimally oxidized LDL (mmLDL) stimulated PI3K activation, Akt, and ERK1/2 phosphorylation, and induced mRNA and protein expression of MIP-2, MCP-1, TNF-, and IL-6 in macrophages. MIP-2 but not MCP-1 secretion depended on TLR4/MyD88, ERK1/2, and PI3K signaling. Thus, mmLDL activates TLR4-dependent and independent proinflammatory signaling in macrophages.

Key Words: macrophage • oxidized low-density lipoprotein • toll-like receptor 4 • phosphoinositide 3-kinase • cytokines

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