Toll-like receptor 4-dependent and -independent cytokine secretion induced by minimally oxidized low-density lipoprotein in macrophages

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 kappa B ( NF-kappa B) p65 translocation to the nucleus, there was no detectable NF-kappa B activation. However, mmLDL induced early mRNA and protein expression of the cytokines MIP- 2, MCP- 1, tumor necrosis factor-alpha, 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.