BACKGROUND: Activated macrophages contribute to the pathogenesis of inflammatory diseases such as atherosclerosis. Although Notch signaling participates in various aspects of immunity, its role in macrophage activation remains undetermined. METHODS AND RESULTS: To explore the role of Notch signaling in inflammation, we examined the expression and activity of Notch pathway components in human primary macrophages in vitro and in atherosclerotic plaques. Macrophages in culture express various Notch pathway components including all 4 receptors (Notch1 to Notch4). Notch3 selectively increased during macrophage differentiation; however, silencing by RNA interference demonstrated that all receptors are functional. The ligand Delta-like 4 (Dll4) increased in macrophages exposed to proinflammatory stimuli such as lipopolysaccharide, interleukin-1beta, or minimally-modified low-density lipoprotein in a Toll-like receptor 4- and nuclear factor-kappaB-dependent fashion. Soluble Dll4 bound to human macrophages. Coincubation of macrophages with cells that expressed Dll4 triggered Notch proteolysis and activation; increased the transcription of proinflammatory genes such as inducible nitric oxide synthase, pentraxin 3 and Id1; resulted in activation of mitogen-activated protein kinase, Akt, and nuclear factor-kappaB pathways; and increased the expression of Dll4 in macrophages. Notch3 knockdown during macrophage differentiation decreased the transcription of genes that promote inflammation, such as inducible nitric oxide synthase, pentraxin 3, Id1, and scavenger receptor-A. These in vitro findings correlate with results of quantitative immunohistochemistry, which demonstrated the presence of Dll4 and other Notch components within macrophages in atherosclerotic plaques. CONCLUSION: Dll4-triggered Notch signaling may mediate inflammatory responses in macrophages and promote inflammation.