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...

The molecule CD23, a low-affinity receptor for IgE (Fc epsilon R2), is a type II transmembrane molecule expressed on many haemopoietic cell types. CD23 has pleiotropic roles in the control of lymphocyte behaviour, suggesting that CD23 may interact with another ligand in addition to IgE. To identify such a CD23 ligand, we expressed and purified full-length recombinant CD23, incorporated it into fluorescent liposomes and used these as a probe. We report here that fluorescent liposomes carrying CD23 interact specifically with the cell-surface protein CD21, identified as the receptor for Epstein-Barr virus and the complement receptor-2 on B cells, some T cells and follicular dendritic cells. In addition, fluorescent CD23-liposomes were shown to bind to hamster kidney cells (BHK-21) transfected with CD21 complementary DNA. The interaction between fluorescent CD23-liposomes and B cells or CD21-transfected BHK-21 cells was specifically inhibited by anti-CD21 and anti-CD23 monoclonal antibodies. Western blotting analysis revealed that 14C-labelled liposomes carrying CD23, in contrast to anti-CD21 antibodies, reacted with a subtype of CD21 molecules. Triggering of CD21 either with an anti-CD21 antibody or with recombinant soluble CD23 was shown to increase specifically interleukin-4-induced IgE production from blood mononuclear cells. These results demonstrate that the cell-surface protein CD21 is a ligand for CD23 and that the pairing of these molecules may participate in the control of IgE production.

Cyclooxygenase-1 (Cox-1) and Cox-2 convert arachidonic acid to prostaglandin H(2), the precursor of other prostaglandins and thromboxanes, eicosanoids important in vascular pathophysiology. However, knowledge of the expression of cyclooxygenases within atherosclerotic lesions is scant. This study tested the hypothesis that human atheroma and nonatherosclerotic arteries express the two Cox isoforms differentially. Cox-1 mRNA and protein localized on endothelial and medial smooth muscle cells of normal arteries (n = 5), whereas Cox-2 expression was not detectable. In contrast, atheromatous (n = 7) lesions contained both Cox-1 and Cox-2, colocalizing mainly with macrophages of the shoulder region and lipid core periphery, whereas smooth muscle cells showed lower levels, as demonstrated by immunohistochemical and in situ hybridization analysis. Furthermore, microvascular endothelium in plaques showed notable staining for both isoforms. In accord with immunohistochemical studies, Western blot analysis of protein extracts from normal arteries revealed constitutive Cox-1, but not Cox-2, expression. Extracts of atheromatous lesions, however, contained both Cox-1 and Cox-2 protein, detected as two immunoreactive proteins of approximately 70 and 50 kd. Macrophages expressed the short form of Cox-1/-2 constitutively after several days of in vitro culture, rather than the 70-kd protein. These results shed new light on the inflammatory pathways that operate in human atheroma. In particular, the expression of Cox-2 in atheromatous, but not in unaffected, arteries has therapeutic implications, given the advent of selective Cox-2 inhibitors.