Interleukin-18: a potent pro-inflammatory cytokine in atherosclerosis: EXPERT'S PERSPECTIVE

This editorial refers to an article by R. Elhage et al.6published in Cardiovascular Research in 2003. It is accompanied by a retrospective editorial by three of the authors of that original article, R. Elhage et al., pp. 176–180, this issue, as part of this Spotlight on Landmark Papers in Cardiovascular Research. The first report on interleukin-18 (IL-18) in patients with myocardial infarction (MI) triggered a significant interest in understanding the role of this cytokine, widely studied in other types of inflammatory diseases, in atherosclerosis.1 Stable and unstable human carotid atherosclerotic plaques were investigated, and it was found that IL-18 was highly expressed compared with control, normal arteries and that it was mainly localized in plaque macrophages.2 IL-18 receptor (IL-18R) was also up-regulated in plaque macrophages and endothelial cells (ECs), suggesting potential biological effects. Significantly higher levels of IL-18 mRNA were found in symptomatic (unstable) plaques as compared with asymptomatic (stable) plaques.2 Electrotransfer of an expression-plasmid DNA encoding murine IL-18-binding protein (BP) in Apo E−/− mice was shown to prevent atherosclerotic plaque progression, inducing changes in plaque composition [a decrease in macrophages, T cells, cell death, and lipid content and an increase in smooth muscle cell (SMC) and collagen content] and leading to a stable plaque phenotype.3 The expression of functional IL-18 and IL-18R was later reported on isolated ECs, SMCs, and macrophages, with the induction of interferon-γ (IFNγ) expression in SMCs.4 Next, studies in Apo E−/− mice showed that IL-18 administration enhanced atherosclerosis in the mouse ascending aorta and the aortic arch through the release of IFNγ.5 However, the IL-18 knockout proof-of-concept was still missing. The effect of blocking the endogenous production of IL18 upon a background of ongoing atherosclerosis progression remained unknown. This information was provided by the important study reported in the article to which this editorial refers, published in Cardiovascular Research in 2003.6 In their interesting article, by inbreeding IL18−/− mice with ApoE−/− mice, Elhage et al. assessed atherosclerosis development to find the reduced lesion size and lesion composition, with an increased SMC content. These observations were independent of cholesterol and triglyceride levels because lipids were significantly higher in the double knock-out mice in spite of reduced atherosclerotic burden. Results were found to be dependent on the lack of IFNγ release.6 Altogether, these experimental findings supported the role of IL-18 in atherosclerotic plaque development, and suggested that IL-18 enhances atherosclerosis through IFNγ release. The study by Elhage et al.6 highlights the inflammatory component of atherosclerosis and the contribution of T helper 1 (Th1)-triggered immunity in the process. Indeed, IL-18 and IL-12 stimulate naïve CD4+ T cells to differentiate into IFNγ-producing Th1 effector cells.7 The IL-18-deficient mice had reduced IFNγ in their atherosclerotic lesions, in agreement with previous results that had shown how the pro-atherogenic effects of recombinant IL-18 were abrogated in IFNγ-deficient animals.5 Thus, IL-18 aggravates atherosclerosis by inducing IFNγ, which promotes the acceleration of the vascular disease. Plaque characteristics significantly differed between the two groups of animals, with an increased proportion of α-SM-actin+ SMC in the IL-18−/−/ApoE−/− animals vs. the IL-18+/+/ApoE−/− animals, reflecting the reduced IFNγ signalling in the plaque and suggesting that these double-knockout animals had more stable plaques.6 IL-18, a member of the IL-1 cytokine superfamily, was first described in 1989 as an IFNγ-inducing factor by Nakamura et al.8 and was cloned by Okamura et al.9 in 1995. Authors were investigating the mechanism underlying the differentiation of CD4+ T cells into functionally distinct subsets (Th1 and Th2). Messenger RNA was found in Kupffer cells and activated macrophages, and IL-18 participation in the mechanisms of tissue injury in inflammatory reactions was proposed. In few years, IL-18 was thoroughly investigated in different immunological-based pathologies and diseases that are associated with inflammation (Figure 1). It was in 2000 that the first reports on acute MI and congestive heart failure (CHF) patients appeared. Increased serum levels of IL-18 were found in acute MI in correlation with the severity of myocardial damage and in CHF patients.1,10 Diagram of the biological functions of IL-18. This diagram shows the ascribed contributions of IL-18 to biological and pathological processes. IL-18 activity is initiated by ligand binding to the heterodimeric receptor complex composed of two chains of the IL-18R, α and β.11 First, IL-18 binds to the IL-18Rα chain, a member of the IL-1R family that is responsible for extracellular binding, and secondly, non-binding signal-transducing β-chain (IL-18Rβ) is recruited to the complex, forming a high-affinity complex.12 Both chains are required for functional signal transduction that starts by the recruitment of the cytosolic protein MyD88 (myeloid differentiation 88) to the IL-18 complex and autophosphorylation of IRAKs (IL-1R-associated kinases). MyD88 functions as an adapter or anchoring molecule, assisting in the binding of the IRAKs to the complex.13,14 IRAK then dissociates from the IL-18R complex and associates with the adaptor protein TRAF-6 (tumour necrosis factor receptor-associated factor 6), which phosphorylates NIK that in turn phosphorylates IκB (via IKKα and IKKβ).15 Once phosphorylated, IκB is rapidly degraded by the ubiquitin pathway, which liberates NFκB so that it translocates to the nucleus for gene transcription (Figure 2). IL-18 binding to IL-18R also activates protein kinases that phosphorylate serine and threonine residues, targeting the MAP kinase family. The IL-18 biological activity is neutralized by IL-18-binding protein (IL-18BP), a protein with high affinity for IL-18.16,17 IL-18 binding to IL-18BP blocks recognition by the IL-18R complex and further signalling (Figure 2).14,18 Interleukin-18 signalling pathway. IRAK, interleukin-1 receptor-associated kinase; TRAF6, tumour necrosis factor-receptor-associated factor-6; IKK, inhibitor of κ light polypeptide gene enhancer in B-cells kinase; NF-κB, nuclear transcription factor-κB; MAPK, mitogen-activated protein kinases; NLRP3, pyrin-domain-containing NRL. The functional effects of IL-18 have expanded the originally identified effector role on IFNγ production. It enhances T and NK cell maturation, cytokine production, and cytotoxicity (FasL-mediated).18 Toll-like receptors (TLRs), innate immune recognition receptors that share homology with receptors for the cytokines IL-1 and the IL-18 family, have also been related to the NOD-like receptors (NLR) or nucleotide-binding domain leucine-rich repeat-containing family of genes.19 NRLs, although recognized for their ability to initiate immune responses to infectious pathogens, are capable of eliciting inflammatory responses to tissue damage or perturbations that are important in the pathogenesis of non-infectious diseases. The pyrin-domain-containing NRL (NRLP) subfamilies have a proposed inflammasome activity.19,20 The NRLP inflammasomes are primarily caspase-1-activating platforms that induce maturation of pro-cytokines, most notably IL-1β and IL-18 (Figure 2). NRLP3 has been associated with cardiovascular disease.21 The IL-18 precursor is then processed by the activated NRLP3 inflammasome, whose oligomerization is dependent on purinergic receptor (P2X7) activation.22 Regarding the use of IL-18 as a biomarker in patients with cardiovascular disease, controversial findings have been reported. IL-18 was initially found as a strong, independent predictor of cardiovascular mortality in stable and unstable angina patients, regardless of the clinical status at admission,23 and in healthy middle-aged subjects in the PRIME study.24 However, two more recent studies using imaging to evaluate subclinical atherosclerosis have been negative.25,26 Probably these contradictory findings are based on studies assessing different patient and healthy cohorts, introducing variability in the results. The question of whether IL-18 can be used as a biomarker for cardiovascular risk prediction remains open. Although a pure anti-inflammatory therapy has not proved effective so far in treating ischaemic cardiovascular disease, research is ongoing. Atherosclerosis in humans is indeed an inflammatory disease that involves the arterial wall and is characterized by the progressively active accumulation of lipids in the vessel wall.27 Lipid levels are never as high in humans as they are in the mouse ApoE−/− model, therefore the background for the development of atherosclerotic lesions is different. However, excessive internalization of lipids is still a hallmark in human atherosclerosis. Endothelial activation enhances EC permeability, resulting in the triggering of the inflammatory cascade with the expression of cytokines/chemokines and adhesion molecules. These events increase lipid particle accumulation and retention, allowing them to become targets for oxidative and enzymatic modifications. Retained pro-atherogenic lipids enhance selective leucocyte recruitment and attachment and transmigration across the endothelium into the intima.28,29 While SMC numbers decline with the severity of plaque progression, monocytes differentiate into macrophages, a process associated with the up-regulation of pattern-recognition receptors, including scavenger receptors and TLRs, leading to foam cell formation (lipid-laden macrophages).27,30 The front of TLRs, NODs, IL-1, and IL-18 stands at the threshold of the adaptive immune response that accelerates full-blown atherosclerotic disease progression. Methotrexate, antagonists of the TNF superfamily, IL-1Ra, IL-6Ra, leukotriene, chemokines, and even immunization are tentative anti-inflammatory pharmacological approaches being tested to inhibit coronary heart disease.31 Presently, anti-inflammatory effects are provided by the use of statins, LDL-lowering drugs with anti-inflammatory effects, but no anti-inflammatory drug is recommended as yet for atherosclerosis prevention. The success of the strategy of targeting inflammatory mediators for the inhibition of coronary artery disease progression will culminate in a field of research that has provided knowledge and understanding on the pathogenesis of atherosclerosis. Conflict of interest: none declared. This work was supported by grants from CIBERobn CB06/03/0050, PN SAF2010-16549, Red TERCEL and Fundacion Jesus Serra—Fundacion Investigacion Cardiovascular, Barcelona.