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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 15, 2004 as doi:10.1096/fj.04-2426fje. |
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* Institut Fédératif de Recherche Paris VII, INSERM U541, Hôpital Lariboisière, Paris, France;
Department of Cardiology, GKT School of Medicine, King’s College London, UK;
Department of Cardiology, University of Göttingen, Göttingen, Germany;
Serono Pharmaceutical Research Institute, Geneva, Switzerland; and
|| Service de Cardiologie, Hôpital Beaujon, Clichy, France
1Correspondence: INSERM U541, Hôpital Lariboisière, 41 Blvd. de la Chapelle, 75475 Paris Cedex 10. E-mail: ziad.mallat{at}larib.inserm.fr
SPECIFIC AIM
It has been suggested that inflammatory mechanisms play a significant pathophysiological role in heart failure. We hypothesized that interleukin 18 (IL-18), a potent proinflammatory cytokine first identified as the interferon 
(IFN-) -inducing factor, is up-regulated in the myocardium of patients with heart failure; we examined the expression of IL-18, its receptor, IL-18R
, and its endogenous inhibitor IL-18 binding protein (IL-18BP) in the normal or failing human myocardium, and determined the circulating plasma levels of IL-18 in patients with heart failure.
PRINCIPAL FINDINGS
1. Up-regulation of IL-18 and IL-18R mRNA but down-regulation of IL-18BP in the failing myocardium
We first examined expression of IL-18, IL-18R, and IL-18BP mRNA in the normal and failing human heart by real-time quantitative RT-PCR. As shown in Table 1
, we found a significant increase in IL-18R mRNA expression and a profound down-regulation of IL-18BP mRNA in the different heart chambers of patients with ischemic (I) or dilated (D) cardiomyopathy (CM). IL-18 mRNA expression was significantly up-regulated in the ischemic left ventricle and septum but not in the corresponding heart chambers of patients with DCM. The right ventricle showed the most prominent alteration of IL-18/IL-18R/IL-18BP levels.
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To determine whether gene expression was associated with the clinical severity of heart failure, we studied a series of patients with DCM with mild or severe heart failure. Changes in IL-18, IL-18R, and IL-18BP gene expression were similar among patients with mild (EF 39.4±2.2%, NYHA II), and patients with more severe (EF 19.8±7.4%, NYHA III/IV) dilated cardiomyopathy, indicating an early alteration of the IL-18/IL-18BP pathway during heart failure.
2. Activation of IL-18 and up-regulation of its receptor, IL-18R, in the failing ischemic and non-ischemic myocardium
Since mRNA levels of IL-18 may not necessarily reflect the levels of the active processed form of IL-18 protein, we examined the expression of the active form of IL-18 protein in the myocardium by Western blot (Fig. 1
). We found a 2- to 4.5-fold increase in the expression of the active form of IL-18 (18 kDa) in DCM (4.93±0.97, P=0.08) and ICM myocardium (12.44±3.10, P<0.01), respectively, in comparison with controls (2.66±0.45). Pro-IL-18 was highly processed to its active form in both ICM and DCM patients in comparison to controls (Fig. 1)
. We detected a significant increase in myocardial expression of IL-18R (Fig. 1)
in both DCM (8.64±1.13, P<0.05) and ICM myocardium (8.32±1.22, P<0.05) in comparison with controls (4.78±0.99).
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Although IL-18 is per se a proinflammatory cytokine, it may greatly synergize with other factors, particularly IL-12, to promote IFN- production. Therefore, we examined the expression of IL-12 in the failing myocardium (Fig. 1)
. Increased expression of active IL-18 in the failing heart was associated with increased expression of IL-12 p70 heterodimer (0.36±0.06, P=0.04 in DCM, n=8 and 0.37±0.04, P=0.015 in ICM, n=6 compared with 0.23±0.02 in NF, n=8).
3. Immunolocalization of IL-18 and IL-18R in the failing myocardium
Using immunohistochemical techniques, we found that IL-18 was mainly expressed in endothelial cells and macrophages but was also detectable in cardiomyocytes surrounded by an important inflammatory infiltrate. On the other hand, IL-18R was expressed on the surface of cardiomyocytes, macrophages, and endothelial cells of ICM myocardium, but was mainly localized to macrophages and other interstitial cells of DCM myocardium. Staining for IL-18 or IL-18R was barely detectable in the NF myocardium. These results point to an important activation of the IL-18 signaling pathway in the failing human heart.
4. Increased levels of plasma IL-18 in patients with heart failure and relation to mortality
We then examined the presence of IL-18 in the peripheral blood of patients with chronic heart failure at the time of admission for an acute episode of decompensation. We found a marked increase in plasma IL-18 levels in patients with decompensated ischemic (354.73±38.24 pg/mL, P=0.003) or non-ischemic cardiomyopathy (362.70±37.10 pg/mL, P=0.0006) in comparison with patients with no signs of chronic heart failure (126.30±19.78 pg/mL). IL-18 levels remained significantly elevated (320.35±41.36 pg/mL) in the subgroup of 17 patients with non-ischemic heart failure studied at a distance from the acute episode. As expected, high levels of plasma BNP, a marker of heart failure, were found in patients with decompensated ischemic (1219.93±147.68 pg/mL) or non-ischemic cardiomyopathy (1107.33±102.46 pg/mL). There was no correlation between IL-18 and BNP levels (P=0.96).
Follow-up (90 days) was available for 46 of 48 patients. Ten of 46 patients died of cardiac causes (21.74%) during follow-up. Median level of plasma IL-18 was significantly elevated (P<0.05) in patients who subsequently died (400 pg/mL; range, 241–910) than in survivors (295 pg/mL; range, 120–617).
CONCLUSIONS AND SIGNIFICANCE
Studies in the last decade have brought an increasingly consistent body of evidence supporting the concept that inflammatory mechanisms, in addition to classic neurohormonal activation, may be involved in the response of myocardium to injury and may contribute to ventricular remodeling and progression to heart failure.
Our interest in the IL-18 pathway as a potential contributor to the pathophysiology of heart failure is based on several important observations. IL-18 may be produced by inflammatory and vascular cells and is involved in both innate and acquired immunoinflammatory responses. In a model of myocardial ischemia-reperfusion injury in vitro, IL-18 was localized to resident macrophages and endothelial cells and was found to be up-regulated after reperfusion injury and processed to its active form by caspase-1. In addition, it has been shown that IL-18 plasma levels are elevated in patients with decompensated heart failure, but no study had examined a potential alteration of IL-18 pathway in the failing human heart.
In addition to the regulation of IL-18 at the level of mRNA expression, particularly in ischemic patients, we found that the precursor form of IL-18 was expressed in the nonfailing myocardium and was almost completely processed to its active form in the failing heart. IL-18 protein was produced by both endothelial cells and infiltrating macrophages and was also detected in cardiomyocytes of inflammatory regions. Therefore, it seems that the ischemic insult with its associated inflammatory response is a major trigger of IL-18 expression since elevated IL-18 mRNA levels were found only in patients with ICM. However, the processing of IL-18 to its active form was enhanced in the myocardium of both ICM and DCM patients, suggesting that other pathophysiological mechanisms were involved. In this regard, the activation of the caspase cascade, already reported in the myocardium of both ICM and DCM patients, may have contributed to increased IL-18 processing. We also found a significant up-regulation of IL-18R that was detected in macrophages, endothelial cells, and cardiomyocytes, suggesting biological effects. Further studies are necessary to identify the precise mechanisms responsible for IL-18/IL-18R up-regulation and/or activation within the failing myocardium, and the relation between IL-18 up-regulation and the expression of other proinflammatory factors, such as TNF.
Besides its roles in the immune response, IL-18 may aggravate the proinflammatory response within the myocardium through different mechanisms (Fig. 2
), including increased expression of endothelial cell adhesion molecules and production of proinflammatory mediators, such as IL-1ß, IL-8, TNF-, and inducible nitric oxide synthase. These proinflammatory mediators have already been implicated in the modulation of myocardial contractile function and loss of cardiomyocytes. On the other hand, IL-18 up-regulates membrane Fas ligand expression and therefore may contribute to Fas-mediated apoptosis of Fas-expressing cardiomyocytes. This pathway may further induce IL-18 production, and has already been involved in both cardiomyocyte apoptosis and cardiomyocyte arrhythmogenicity related to heart disease. Moreover, IL-18 is emerging as a potent antiangiogenic cytokine and so may severely affect myocardial neoangiogenesis after ischemia with its expected deleterious consequences on myocardial functional recovery and remodeling. Clearly, future studies are necessary to examine in more details the relevance of IL-18 inhibition in the context of heart failure.
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In agreement with the results of a recently published study, we found a significant increase in plasma IL-18 levels in patients with decompensated heart failure of ischemic or non-ischemic origin, in comparison with patients with no heart failure. Plasma IL-18 levels measured in the present study were not correlated to BNP levels, suggesting that they reflect different pathophysiological mechanisms. In addition, we observed a greater and significant increase in IL-18 levels in the patients who died during follow-up than in survivors, suggesting a potential prognostic and mechanistic role for IL-18 in heart failure. These important issues merit further investigation.
Our study provides the first evidence for an up-regulation of the immunoinflammatory IL-18 pathway in the failing heart and an association with disease severity. We believe that these findings are important for several reasons. 1) IL-18 has already been shown by several groups, including ours, as a critical proatherogenic and plaque-destabilizing factor, strongly related to cardiovascular mortality in coronary patients. 2) IL-18 is unique among the proinflammatory mediators in that it exerts potent antiangiongenic rather than proangiogenic activity in the context of ischemic injury. Therefore, it is expected that therapeutic strategies aiming at IL-18 inhibition in ischemic diseases and/or heart failure would also enhance neo-angiogenesis (as we have previously shown in murine models), with potential additional benefits on tissue remodeling. 3) IL-18 may act downstream of TNF and inhibition of IL-18 in experimental models of tissue injury has been shown to be protective despite persistent high levels of TNF, suggesting that our findings of increased IL-18 expression and/or processing in the failing human heart are per se worth consideration, whatever the levels of TNF production.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2426fje;
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