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(Circulation. 1997;96:1386-1389.)
© 1997 American Heart Association, Inc.

Articles

Amiodarone Inhibits Production of Tumor Necrosis Factor- by Human Mononuclear Cells

A Possible Mechanism for its Effect in Heart Failure

Akira Matsumori, MD, PhD; Koh Ono, MD; Ryosuke Nishio, MD; Yoshisuke Nose, PhD; ; Shigetake Sasayama, MD, PhD

From the Department of Cardiovascular Medicine, Kyoto University, Japan.

Correspondence to Akira Matsumori, MD, PhD, Department of Cardiovascular Medicine, Kyoto University, 54 Kawaracho Shogoin, Sakyo-ku, Kyoto 606, Japan. E-mail amat{at}kuhp.kyoto-u.ac.jp

	Abstract

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Background Recent studies suggest that cytokines such as tumor necrosis factor (TNF)- and interleukins (ILs) are capable of modulating cardiovascular function and that drugs used in the treatment of heart failure have various modulatory effects on the production of cytokines. This study was performed to examine the effects of amiodarone (a drug shown to be beneficial in some patients suffering from heart failure) versus other antiarrhythmic agents on the production of cytokines in vitro.

Methods and Results Human peripheral blood mononuclear cells (PBMC) were obtained from healthy volunteers. PBMC were cultured with 0.1, 1, and 10 µmol/L of amiodarone, quinidine, disopyramide, and lidocaine in the presence of lipopolysaccharide. After 24 hours' incubation, TNF-, IL-1ß, and IL-6 were measured in the culture supernatants by an enzyme-linked immunosorbent assay. TNF- production was inhibited by amiodarone but stimulated by quinidine in a concentration-dependent manner. Disopyramide and lidocaine tended to increase TNF- production. IL-6 production was decreased by amiodarone in all concentrations but was increased significantly by disopyramide. Modulation of IL-1ß production by amiodarone was biphasic and significantly increased at a concentration of 10 µmol/L.

Conclusions These previously unrecognized immunomodulatory effects of amiodarone may contribute to its beneficial effects in heart failure patients.

Key Words: amiodarone • heart failure • immune system • interleukins

	Introduction

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With the growing use of amiodarone for the treatment of arrhythmias in patients with CHF, a concomitant improvement was noted in measurements of left ventricular function^{1 2} and exercise tolerance.² Although amiodarone did not reduce all-cause or sudden death mortality,³ the substantial reduction in the combined end point of cardiac death plus hospitalization for heart failure in patients with nonischemic disease is noteworthy.^{3 4} These apparent benefits are in marked contrast to the negative inotropic and hemodynamic responses to other antiarrhythmic agents in this patient population^{5 6} and, along with a low incidence of proarrhythmic events,⁷ have made amiodarone the antiarrhythmic agent of choice in the treatment of CHF patients.^{6 7 8}

Recent studies from our laboratory have shown that drugs for heart failure modulate the production of cytokines in various ways^{9 10 11} and suggest that some of these immunomodulatory effects are relevant to the effects of drugs in heart failure patients. Accordingly, the present study was performed to investigate whether the positive hemodynamic effects of amiodarone might be attributable to a modulation of cytokine production.

	Methods

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PBMC were obtained from healthy volunteers and collected by Ficoll-paque (Pharmacia) density centrifugation. The cells were washed three times with PBS, resuspended in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated fetal calf serum (Gibco), 100 U/mL penicillin, 100 µg/mL streptomycin (Gibco), and 50 µmol/L 2-mercaptoethanol, and cultured at 37°C in a humidified 5% CO₂ atmosphere. Amiodarone, quinidine, disopyramide and lidocaine (all from Sigma Chemical Co) were dissolved in 0.1% DMSO and diluted with the medium. This concentration of DMSO did not influence the production of cytokines by PBMC. PBMC (2x10⁶ cells/mL in 24-well plates) were stimulated with 1 µg/mL of LPS, to which 0.1, 1, and 10 µmol/L of each drug was added. After 24 hours' incubation, the supernatants were harvested and stored at -80°C until performance of the cytokine assay. TNF-, IL-1ß, and IL-6 levels in the culture supernatants were determined by specific enzyme-linked immunosorbent assay kits (Otsuka Pharmaceutical Co). Because significant interindividual variability of cytokine production by PBMC in response to LPS was observed, cytokine production from two to five subjects was measured (2 wells from each). None of the agents affected monocyte viability, verified by staining with trypan blue.

Statistical Analyses
Because the amount of the cytokines produced was different in each experiment, the effects of the drugs on cytokine production was expressed as a percent response compared with the control wells containing LPS. Statistical analyses were performed by one-way ANOVA with multiple comparisons by Fisher's protected least significant difference test. All values are presented as the mean±SE. A value of P<.05 was considered statistically significant.

	Results

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TNF- production was decreased by amiodarone in a concentration-dependent manner. Compared with a stimulated control of 2.15±0.57 ng/mL, concentrations of 0.1, 1, and 10 µmol/L (n=10 each) decreased TNF- by 70±15%, 61±13%, and 54±10%, respectively (P<.05 at 1 µmol/L, P<.01 at 10 µmol/L versus control; Fig 1). In contrast, quinidine increased TNF- production in a concentration-dependent manner. Compared with a stimulated control of 6.69±3.3 ng/mL, quinidine concentrations of 0.1, 1, and 10 µmol/L (n=4 each) increased TNF- by 117±10%, 131±7%, and 149±13%, respectively (P<.05 and P<.01 at 1 and 10 µmol/L, respectively; Fig 1). Compared with a stimulated control of 0.86±0.07 ng/mL, disopyramide increased the production of TNF- at all concentrations (162±30%, 151±20%, and 147±33%, respectively; n=4 each). However, these differences did not reach statistical significance.

View larger version (51K): [in this window] [in a new window]   Figure 1. Effects of amiodarone, quinidine, disopyramide, and lidocaine on the production of TNF- by PBMC stimulated with LPS. *P<.05; P<.01 vs control.

IL-6 production was significantly inhibited by amiodarone at all three concentrations (67±6%, 65±6%, and 66±3%; P<.0001) compared with a stimulated control value of 2.1±0.4 ng/mL (Fig 2). Conversely, in comparison with a stimulated control of 1.7±0.2 ng/mL, disopyramide increased IL-6 production significantly (120±5%, 113±3%, and 122±5%, respectively; P<.05) whereas quinidine and lidocaine had no significant effect (Fig 2).

View larger version (40K): [in this window] [in a new window]   Figure 2. Effects of amiodarone, quinidine, disopyramide, and lidocaine on the production of IL-6 by PBMC stimulated with LPS. *P<.05; P<.01; P<.0001 vs control.

IL-1ß in the presence of amiodarone was slightly decreased at concentrations of 0.1 and 1 µmol/L (76±11% and 68±9%, respectively, versus a stimulated control of 8.6±3.8 ng/mL) but markedly increased at a concentration of 10 µmol/L (228±72%; P<.05) (Fig 3). IL-1ß production was not significantly changed by quinidine, disopyramide, or lidocaine.

View larger version (15K): [in this window] [in a new window]   Figure 3. Effects of amiodarone, quinidine, disopyramide, and lidocaine on the production of IL-1ß by PBMC stimulated with LPS. *P<.05.

	Discussion

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Several studies have measured an improvement in left ventricular ejection fraction or indices of clinical heart failure with amiodarone therapy.^{1 2 12} This favorable effect is particularly apparent in patients suffering from nonischemic heart disease.⁴ Modulation of cardiovascular function by cytokines has been well documented.¹³ Elevated concentrations of TNF- have been reported in patients with chronic heart failure,^{14 15} and TNF- has been reported to depress myocardial contractility.^{16 17 18 19} Furthermore, TNF-, IL-1ß, and interferon- have cytotoxic effects on cultured cardiac myocytes.²⁰ In addition to these humoral effects, cytokines may activate cytotoxic T-cells, thereby causing direct injury to the myocyte.²¹ IL-6 may also exert a negative inotropic effect,¹⁶ and mice overexpressing both IL-6 and IL-6 receptors have been reported to develop cardiac hypertrophy.²² Other recent observations suggest that growth abnormalities, which accompany hypertrophy of the overloaded myocardium, may play an important role in the deterioration of the condition of patients with chronic heart failure.²³

Recent studies from our laboratory¹⁴ have shown that increased concentrations of circulating TNF- are found more often in patients with nonischemic than those with ischemic heart failure. In our animal models of nonischemic heart failure induced by viral myocarditis,^{24 25} plasma TNF- concentration was increased in the blood, and exogenously given anti–TNF- antibody improved both myocardial injury and survival of the animals.²⁶ Furthermore, in the same model, the expression of TNF- messenger RNA was increased in the acute stage and persisted into the chronic stage,²⁷ and immunohistochemical analyses revealed that endothelial cells and interstitial macrophages were positive for TNF-. These observations suggest that TNF- plays an important role in the pathogenesis of nonischemic heart failure.

The reduced production of TNF- and IL-6 by amiodarone measured in the present study was in sharp contrast to the effects produced by three other antiarrhythmic agents and may explain the hemodynamic effects of amiodarone noted in clinical studies. Therapeutic plasma concentrations of amiodarone range between 0.5 and 2 µg/mL, which is equivalent to 0.87 to 3.1 µmol/L, within the range of concentrations used in these experiments.

The mechanisms by which amiodarone modulates cytokine production remain to be clarified. The involvement of ion channels in T-lymphocyte activation and proliferation is well established.^{28 29} The involvement of K⁺ channels in lymphocyte activation and cytotoxic function has been reported recently with the use of the scorpion-derived peptide margatoxin, a specific K⁺ channel blocker in lymphocytes.³⁰ Vesnarinone, which has been reported to reduce K⁺ current,³¹ inhibits the production of cytokines.¹⁰ Because amiodarone blocks the inward rectifier K⁺ channel,³² it may inhibit cytokine release by modulating ion channels. This hypothesis, if verified, may help in the development of more targeted pharmacological treatments of CHF.

Additional studies are necessary to confirm our findings using lymphocytes from patients with CHF. Our observations do not establish a direct connection between the effects of amiodarone on cytokines and its hemodynamic effects. Additional studies will be needed to examine this link in vivo in animal models, as well as by measuring cytokines in patients suffering from CHF who are treated with amiodarone.

	Selected Abbreviations and Acronyms

 

CHF = congestive heart failure IL = interleukin LPS = lipopolysaccharide PBMC = peripheral blood mononuclear cells TNF = tumor necrosis factor

	Acknowledgments

 
We would like to thank Y. Ohmoto and H. Toriyama for their assistance in the assay of cytokines and T. Nakano and S. Sakai for preparing the manuscript.

Received April 16, 1997; revision received June 4, 1997; accepted June 10, 1997.

	References

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