β-Adrenergic stimulation induces interleukin-18 expression via β2-AR, PI3K, Akt, IKK, and NF-κB

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Abstract

We investigated whether β-adrenergic receptor (β-AR) stimulation induces the expression of interleukin (IL)-18, a proinflammatory cytokine, in myocardium and in cardiac-derived endothelial cells (CDEC) via activation of nuclear factor (NF)-κB. Our results indicate that isoproterenol (ISO) activates NF-κB DNA binding activity, and induces myocardial and systemic elaboration of IL-18 via β2-AR signaling. Furthermore, in CDEC, ISO increased basal and inducible promoter activities, increased IL-18 gene transcription and mRNA stability, and induced IL-18 expression via β2-AR agonism. Signaling required Gi, PI3K, Akt, IKK, and NF-κB. In conclusion, our results indicate for the first time that isoproterenol induces myocardial and systemic elaboration of IL-18 via a β2-AR and NF-κB-dependent mechanism. Similar events may occur in heart failure, a disease state characterized by sustained β-AR activation.

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Materials and methods

Animals. All studies were performed in compliance with the NIH Guide for the Care and Use of Laboratory Animals (DHHS Publication No. [NIH] 85-23, revised 1996). Male C57Bl/6 mice weighing 25–30 g (Charles River) were anesthetized with IM ketamine (30 mg/kg), acepromazine (1 mg/kg), and xylazine (6 mg/kg) before subcutaneous implantation of miniosmotic pumps (1.0 μL/h; Alzet, model 2001; DURECT, Cupertino, CA) for a continuous infusion of either l-isoproterenol [(−)-isoproterenol hydrochloride,

β-AR stimulation induces myocardial and systemic elaboration of IL-18

Administration of ISO increased NF-κB DNA binding activity in left ventricular tissue with peak levels detected at 2 h (Fig. 1A; 6.72-fold, p<0.01 versus saline). A supershift was observed with both anti-p50 and anti-p65 subunit-specific antibodies (Fig. 1B). As compared to saline controls, ISO-induced IL-18 mRNA expression in a rapid and sustained manner for up to 72 h (Fig. 1C; 2 h, 3.21-fold; 3 h, 4.23-fold; both p<0.025). Western blotting revealed increased IL-18 protein levels following ISO

Discussion

We have shown for the first time that treatment of mice with ISO results in IL-18 expression in myocardium via β2-AR and NF-κB activation. Similarly in isolated cardiomyocytes, addition of ISO induces IL-18 expression via β2-AR stimulation. Furthermore, ISO induces IL-18 expression in CDEC through β2-AR stimulation, Gi, PI3Kγ, Akt, IKK, and NF-κB, activates both basal and inducible IL-18 promoter activities, and upregulates IL-18 mRNA expression via increased gene transcription and mRNA

Acknowledgements

This work was supported in part by National Institutes of Health Grant HL68020 to B.C. S.D.P. is supported by a Veterans Administration merit grant and NIH PO1 ES011860-01A1. We thank Dr. Gregory L. Freeman for his helpful discussions and criticism of the manuscript.

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