Intracellular Function of Interleukin-1 Receptor Antagonist in Ischemic Cardiomyocytes

Background: Loss of cardiac myocytes due to apoptosis is a relevant feature of ischemic heart disease. It has been described in infarct and peri-infarct regions of the myocardium in coronary syndromes and in ischemia-linked heart remodeling. Previous studies have provided protection against ischemia-induced cardiomyocyte apoptosis by the anti-inflammatory cytokine interleukin-1 receptor-antagonist (IL-1Ra). Mitochondria triggering of caspases plays a central role in ischemia-induced apoptosis. We examined the production of IL-1Ra in the ischemic heart and, based on dual intra/extracellular function of some other interleukins, we hypothesized that IL-1Ra may also directly inhibit mitochondria-activated caspases and cardiomyocyte apoptosis. Methodology/Principal Findings: Synthesis of IL-1Ra was evidenced in the hearts explanted from patients with ischemic heart disease. In the mouse ischemic heart and in a mouse cardiomyocyte cell line exposed to long-lasting hypoxia, IL-1Ra bound and inhibited mitochondria-activated caspases, whereas inhibition of caspase activation was not observed in the heart of mice lacking IL-1Ra (Il-1ra−/−) or in siRNA to IL-1Ra-interfered cells. An impressive 6-fold increase of hypoxia-induced apoptosis was observed in cells lacking IL-1Ra. IL-1Ra down-regulated cells were not protected against caspase activation and apoptosis by knocking down of the IL-1 receptor, confirming the intracellular, receptor-independent, anti-apoptotic function of IL-1Ra. Notably, the inhibitory effect of IL-1Ra was not influenced by enduring ischemic conditions in which previously described physiologic inhibitors of apoptosis are neutralized. Conclusions/Significance: These observations point to intracellular IL-1Ra as a critical mechanism of the cell self-protection against ischemia-induced apoptosis and suggest that this cytokine plays an important role in the remodeling of heart by promoting survival of cardiomyocytes in the ischemic regions

Effetto antiapoptotico dell' IL-1ra nei miocardiociti ischemici-l'endotelio linfatico nella ricircolazione leucocitaria.

2006/2007EFFETTO ANTIAPOPTOTICO DELL’IL-1ra NEI MIOCARDIOCITI ISCHEMICI La morte di cellule cardiache per apoptosi è una fenomeno rilevante nelle cardiomiopatie ischemiche. Questo fenomeno è stato descritto nelle regioni peri-infartuali del miocardio nelle patologie coronariche e nel rimodellamento cardiaco dovuto a patologie ischemiche. L’attivazione, per via mitocondriale, della caspasi-9 gioca un ruolo fondamentale nell’apoptosi indotta da ischemia. Con questo lavoro si vuole dimostrare che: - l’antagonista recettoriale dell’interleuchina-1 (IL-1ra), che è catalogata come una citochina infiammatoria, è presente nei sieri di pazienti con una sindrome coronarica acuta a concentrazioni elevate. - l’IL-1ra è attivamente prodotta dalle cellule cardiache ischemiche. - l’IL-1ra inibisce, in vitro, l’attività della caspasi-9. - l’IL-1ra è una molecola che, in vivo, fa preservare i miocardiociti ischemici dalla morte per apoptosi. La scoperta di questa nuova funzione endogena dell’IL-1ra evidenzia l’importanza di questa proteina nel rimodellamento cardiaco, promuovendo la sopravvivenza dei miocardiociti nelle regioni ischemiche. Queste osservazioni pongono le basi razionali per iniziare a studiare possibili interventi terapeutici in specifiche patologie dove, con il controllo dell’apoptosi attivata per via mitocondriale, si potrebbero ottenere dei vantaggi sul piano clinico. L’ENDOTELIO LINFATICO NELLA RICIRCOLAZIONE LEUCOCITARIA La presente ricerca ha come obiettivo quello di mettere in evidenza il ruolo dell'endotelio dei vasi linfatici nella cosiddetta ricircolazione dei leucociti e nell’entrata nel circolo linfatico di cellule tumorali maligne in fase di metastatizzazione. Più precisamente il nostro obiettivo è quello di identificare quali chemochine prodotte dalle cellule endoteliali linfatiche siano implicate nella migrazione cellulare. Dopo aver confermato l’espressione di alcune chemochine da parte dell’endotelio linfatico, in particolare della C10, si è individuata la possibilità di costruire grazie ad una tecnica di silenziamento genico, l’RNAi, modelli sperimentali in cui le cellule con attività chemiotattica nei confronti delle cellule endoteliali linfatiche siano deficitarie dei recettori per la chemochina C10. Si è osservato che nelle cellule trattate diminuisce l’espressione dei recettori, confermando l’efficacia della tecnica di silenziamento genico utilizzata. Si è anche valutata la capacità delle cellule trattate di migrare verso un gradiente chemiotattico prodotto dalle cellule endoteliali linfatiche, constatando che le cellule trattate, dunque deficitarie del recettore, mostrano una minore capacità migratoria nei confronti del terreno condizionato dalle cellule endoteliali linfatiche. Si è anche constatato che, in vivo, le cellule deficitarie per i recettori della C10 hanno minore capacità di infiltrare il tessuto linfatico. Questi risultati convalidano la tesi che la chemochina C10 sia implicata nel processo di migrazione. Gli esperimenti precedentemente descritti sono sempre stati condotti su cellule murine; fino ad ora infatti esiste solo la possibilità di ottenere colture endoteliali linfatiche murine. Per poter studiare tali fenomeni nell’uomo si sta cercando di isolare cellule endoteliali linfatiche umane. A tale scopo si è prodotto in cavia un anticorpo che fosse specifico per la podoplanina umana. La podoplanina è una mucoproteina di membrana espressa dall’endotelio linfatico e non da quello vascolare, quindi costituisce un valido marcatore per distinguere questi due tipi di endotelio.XX Cicl

IL-1R1-independent anti-apoptotic function of IL-1Ra.

<p>(a) Immunofluorescence of IL-1Ra (green) and nuclear DAPI (blue) staining of cultured mouse cardiomyocytes (HL-1 cells) incubated for 6 hr in normoxia (panel i), or hypoxia (95%N₂-5%C0₂ panel ii) conditions, and (b) rate of IL-1Ra positive cells (%) in fig. a conditions. (c) Double-immunofluorescence for IL-1Ra and IL-1R1 (both green, panel i), or (d) for IL-1Ra (green, panel i), or (e) for IL-1R1 (green, panel i), or (f) for IL-1Ra and IL-1R1 (both green, panel i), respectively, together with TUNEL co-staining (red, panel ii) in the same field (merge, panel iii) of cultured cardiomyocytes treated with siRNA to both IL-1Ra and IL-1R1, or to IL-1Ra alone, or to IL-1R1 alone, or with control siRNA, respectively, and exposed to 6 hr hypoxia. Bars, 20 um. (g) Rate of TUNEL positive cells (%) in fig. c-f conditions. Results are means ± SE, and were obtained using three siRNA probes to IL-1Ra. n = 8. *p<0.001 vs controls. (h) Western blot detection of IL-1Ra and IL-1R1 protein expression in fig. c-f conditions. (i) RTqPCR analysis of IL-6 mRNA expression in HL-1 cardiomyocytes treated with siRNA to both IL-1Ra and IL-1R1, or control siRNA, and cultured for 5 hr in the presence or absence of IL-1 beta ((40 pg/ml) or TNF alpha (10 ng/ml), corrected for mRNA expression of beta-actin. The results confirm down regulation of the IL-1 receptor (IL-R1) in siRNA-treated HL-1 cells. The bars show mean ± SE of four experiments; *p<0.001 vs activity of TNF alpha-treated controls.</p

Expression of IL-1Ra in hearts explanted from patients with end stage ischemic heart disease.

<p>(a) Immunofluorescence co-staining for IL-1Ra and PECAM-1. Several cardiomyocytes show positive staining for IL-1Ra (green), whereas PECAM-1 positive (red) endothelial cells of myocardial microvessels do not co-stain with IL-1Ra. Cell nuclei are evidenced by DAPI (blue) stain. (b) Co-staining of IL-1Ra (brown) and fibroblast specific vimentin (red), and (c) of IL-1Ra (brown) and leukocyte/macrophage specific CD14 (red). Nuclei are lightly counterstained by Mayer’s Hematoxylin. (d) In situ hybridization for IL-1Ra mRNA. Several cardiomyocytes stained positive for the in situ hybridization in a large area of peri-infarct scar viable myocardium. The inset shows how in situ hybridization is localized mainly in perinuclear areas within cardiomyocytes. (e) Co-staining for IL-1Ra and active caspase3 in a peri-infarct scar area. Besides IL-1Ra positive cardiomyocytes (brown) there are several caspase3-positive cells (red). Bars: a–c, e 20 um, d 40 um, insets 10 um. (f) qRT-PCR analyses of sIL-1Ra and icIL-1Ra (type-1, and type-3) mRNA in ischemic cardiomyopathy, corrected for mRNA expression of β-actin. The graph compares heart regions with macroscopic features of normal blood supply and trophism (remote) to heart areas close to post infarct scars (peri infarct-scar) and regions 1 cm away from the scars (intermediate). The bars show mean ± SE of five experiments.</p

IL-1Ra protects cardiomyocytes from ischemia-induced apoptosis.

<p>(a) Hystochemistry of IL-1Ra expression (purple) in the heart following coronary artery ligation in mice: ventricle cross section, and (b) specific, diffuse IL-1Ra staining of cardiomyocytes in the ischemic heart area. (c) Time course of secreted (s) and intracellular (ic) IL-1Ra mRNA expression in the hypoxic heart of WT (Il-1ra+/+) mice. The graphs represent the fold change after normalization with the expression of β-actin. (d) Histology of TUNEL staining (red stain) of Il-1ra+/+ and (e) Il-1ra−/− mouse hearts after 6 hr hypoxia, and of (f) Il-1ra+/+ and (g) Il-1ra−/− mouse hearts not exposed to hypoxia. (h) Rate of TUNEL staining in d-g conditions. Results are means ± SE, n = 3, **p<0.001 for Il-1ra−/− vs control Il-1ra+/+ mouse hearts after 6 hr hypoxia, *p<0.001 for Il-1ra+/+ mouse hearts after 6 hr hypoxia vs hearts not exposed to hypoxia. Bars, a 2 mm, b 20 um; d, e, g, h 40 um.</p

Coimmunoprecipitation of IL-1Ra with mitochondria-activated caspases.

<p>(a) Coimmunoprecipitation of IL-1Ra with caspase-9 and (b) with caspase-3, -6, and -7 in cultured HL-1 cardiomyocytes after 6 hr hypoxia. Detection by western blot with monoclonal Abs to caspases or to IL-1Ra, or to control proteins IL-1beta, IL-1 type I receptor (IL-1R1) and IL-1R Ancillary Protein (IL-1R AcP). Proteins immunoprecipitated (IP) by Abs to caspases or to IL-1Ra, or to IL-1beta (control) are compared to unbound (free) supernatant proteins. The data are compiled from different gels in three separate experiments; [ ] not detected.</p

Hypothetical model of IL-1Ra in the inhibition of apoptosis.

<p>Smac released from ischemia-induced mitochondria which inhibits the neutralizing effect of IAPs on caspases. Ischemia-induced IL1ra interacts with caspase-9 and blocks cell death.</p

In vitro activity of terminal caspases in cardiomyocytes lacking IL-1Ra.

<p>Activity of mitochondria-activated terminal caspases in cytosols of cultured HL-1 cardiomyocytes untreated or treated with siRNA to IL-1Ra RNA, or both IL-1Ra and IL-1R1 RNAs, and then incubated for 6 hr in normoxia or hypoxia conditions. Ac-DEVD-AMC assays compare enzyme activity in the absence (controls) or presence of anti-IL-1Ra Abs. Bars show means ± SE of 3 exp.; *p<0.01 vs activity of controls.</p