Background-Interleukin (IL)-18 is a potent proinflammatory cytokine with potential atherogenic properties. Its expression and role in atherosclerosis, however, are unknown. Methods and Results-In the present study, we examined stable and unstable human carotid atherosclerotic plaques retrieved by endarterectomy for the presence of IL-18 using reverse transcription-polymerase chain reaction (PCR), Western blot, and immunohistochemical techniques. IL-18 was highly expressed in the atherosclerotic plaques compared with control normal arteries and was localized mainly in plaque macrophages. IL-18 receptor was also upregulated in plaque macrophages and endothelial cells, suggesting potential biological effects. To examine the role of IL-18 in atherosclerosis, we determined the relation between IL-18 mRNA expression and signs of plaque instability using real-time quantitative PCR. Interestingly, significantly higher levels of IL-18 mRNA were found in symptomatic (unstable) plaques than asymptomatic (stable)

Abstract-Interleukin (IL)-18 is the interferon-␥-inducing factor and has other proinflammatory properties. The precise role of IL-18 in immunoinflammatory diseases remains poorly understood. In this study, we show that in vivo electrotransfer of an expression-plasmid DNA encoding for murine IL-18 binding protein (BP) (the endogenous inhibitor of IL-18) prevents fatty streak development in the thoracic aorta of apoE knockout mice and slows progression of advanced atherosclerotic plaques in the aortic sinus. More importantly, transfection with the IL-18BP plasmid induces profound changes in plaque composition (decrease in macrophage, T cell, cell death, and lipid content and increase in smooth muscle cell and collagen content) leading to a stable plaque phenotype. These results identify for the first time a critical role for IL-18/IL-18BP regulation in atherosclerosis and suggest a potential role for IL-18 inhibitors in reduction of plaque development/progression and promotion of plaque stability. Key Words: atherosclerosis Ⅲ inflammation Ⅲ interleukin Ⅲ cytokines A therosclerosis is the leading cause of mortality in industrialized countries and carries an important socioeconomic burden. Unabated inflammatory mechanisms are responsible for changes in atherosclerotic plaque composition leading to plaque disruption and to the occurrence of acute ischemic syndromes, namely myocardial infarction and stroke. 1 Interleukin (IL)-18 is an inducer of interferon (IFN)-␥ with potent activities on inflammatory and vascular cells 2 and is thought to contribute to the pathogenesis of chronic immunoinflammatory processes. 3,4 Interestingly, we have recently detected increased production of IL-18 by macrophages and smooth muscle cells in unstable human atherosclerotic plaques that were responsible for strokes compared with stable plaques from asymptomatic patients. 5 An endogenous IL-18 binding protein (IL-18BP) that neutralizes IL-18 has been identified. 6 However, the role of IL-18BP in the modulation of atherogenesis and other chronic immunoinflammatory diseases in vivo is currently unknown. In this study, we examined the role of IL-18/IL-18BP in a wellvalidated model of atherosclerosis. Materials and Methods In Vivo Intramuscular Electrotransfer of Murine IL-18BP Expression PlasmidFourteen male C57BL/6 apoE knockout mice, 14 weeks old, received at 3-week intervals, 3 injections with the murine IL-18BP expression plasmid, pcDNA3-mIL18BP. The control mice (nϭ19) were injected with the control empty plasmid. Murine IL-18BP isoform d cDNA (accessory number #AF110803), isolated as described, 6 was subcloned into the EcoR1/Not1 sites of mammalian cell expression vector pcDNA3 under the control of the cytomegalovirus promotor (Invitrogen). Control vector was a similar construct devoid of therapeutic cDNA. The construct with mIL-18BP isoform d in pCDNA3 plasmid was tested for expression and activity. This was performed using culture supernatants obtained from HEK 293/Ebna cells transfected with mIL-18BPd in pCDNA3 vector. We verified...