Enhanced production of TNF-alpha from macrophages promotes development and instability of atherosclerotic plaques, but involvement of lipid component in TNF-alpha production has not been clarified in atherosclerosis. We attempted to determine whether cholesterol oxidation products (oxysterols) could modify TNF-alpha production. Treatment of THP-1 cells with 27-hydroxycholesterol (27OHChol) or 7 alpha-hydroxycholesterol (7 alpha OHChol) resulted in a profound increase in TNF-alpha, transcription, while treatment with an identical concentration of cholesterol and 7-ketochoelsterol did not lead to any change in TNF-alpha expression. Treatment with 27OHChol resulted in increased synthesis, as well as secretion, of TNF-alpha, while 7 alpha OHChol led to increased synthesis of TNF-alpha without affecting secretion of the cytokine. Co-treatment with 7 alpha OHChol or 27OHChol and LPS resulted in synergistically enhanced or augmented secretion of TNF-alpha. Treatment with TO-901317, pertussis toxin, PP2, and LY294002 resulted not only in attenuated transcription of TNF-alpha induced by 27OHChol and 7 alpha OHChol, but also secretion of TNF-alpha enhanced by 27OHChol. This is the first report demonstrating enhanced production of TNF-alpha in macrophages by treatment with oxysterols which are detected in abundance in atheromatous lesions; in addition, results of the current study provide evidence indicating that certain types of oxysterols contribute to development of atherosclerosis by promoting production of proinflammatory cytokines. (C) 2012 Elsevier Inc. All rights reserved.