An agonist of adenosine A3 receptors decreases interleukin-12 and interferon-γ production and prevents lethality in endotoxemic mice

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Abstract

We have recently observed that the selective adenosine A3 receptor agonist N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) augments interleukin-10 and inhibits tumor necrosis factor-α production in endotoxemic mice. In the present study, we extended our investigations into the effect of this compound on the bacterial lipopolysaccharide (endotoxin)-induced inflammatory response in the BALB/c, as well as in the C57BL/6 interleukin-10+/+ and the interleukin-10 deficient C57BL/6 interleukin-100/0 mice strains. In the BALB/c mice, i.p. pre-treatment with IB-MECA (0.2 and 0.5 mg/kg) decreased lipopolysaccharide (60 mg/kg i.p.)-induced plasma levels of interleukin-12 (p40 and p70), interferon-γ, and nitrite/nitrate (breakdown products of nitric oxide (NO)). On the other hand, pre-treatment with this compound failed to influence lipopolysaccharide-induced plasma interleukin-1α, interleukin-6, and corticosterone concentrations. Similar to its effect in BALB/c mice, IB-MECA enhanced the release of interleukin-10 in the C57BL/6 interleukin-10+/+ mice. Furthermore, IB-MECA inhibited the production of interleukin-12, interferon-γ, and NO in both the C57BL/6 interleukin-10+/+ and C57BL/6 interleukin-100/0 mice, suggesting that the inhibition of pro-inflammatory cytokine production by this compound is independent of the increased release of interleukin-10. Finally, pre-treatment with this compound protected mice against lipopolysaccharide (60 mg/kg i.p.)-induced lethality. These results indicate that stimulation of adenosine A3 receptors has potent anti-inflammatory effects and may represent a potential strategy in the treatment of septic shock and other inflammatory diseases.

Introduction

The sequential release of pro-inflammatory mediators in response to bacterial, viral, or fungal infections is essential in the fight against the invading microorganisms. However, the overproduction of these mediators may be detrimental for the host leading to multi-organ failure, shock, and finally death. The systemic administration of bacterial lipopolysaccharide (endotoxin), a cell-wall component of Gram-negative bacteria is a prototypic stimulus for the activation of an inflammatory cascade. Early in the course of this cascade appear the monocyte/macrophage derived pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1, and interleukin-12, which play a key role in the pathogenesis of endotoxic shock (Beutler, 1995; Trinchieri, 1995). These cytokines are central to the induction of interferon-γ production by natural killer cells and T lymphocytes (D'Andrea et al., 1993; Heinzel et al., 1994; Wysocka et al., 1995), which is another important intermediate in the development of endotoxin shock (Doherty et al., 1992). Tumor necrosis factor-α, interleukin-1, and interferon-γ trigger the subsequent induction of the macrophage type inducible nitric oxide (NO) synthase (Szabó et al., 1993; Thiemermann et al., 1993; Salkowsky et al., 1997) leading to the overproduction of NO. Formation of this free radical, or the related toxic oxidant product peroxynitrite crucially contribute to the development of hypotension, vascular hyporeactivity, endothelial injury, and mortality in endotoxin shock (Szabó, 1995). Interleukin-10, which is also released in the early phases of endotoxemia, inhibits the production of tumor necrosis factor-α, interleukin-12, interferon-γ, and NO (Berg et al., 1995; Haskó et al., 1998a), and is protective in both lipopolysaccharide- and staphylococcal enterotoxin B-induced lethality (Berg et al., 1995; Haskó et al., 1998b).

Adenosine is a purine nucleoside that is released from cells in response to metabolic stress (Dubyak and El-Moatassim, 1993) or from the sympathetic nervous system (White and MacDonald, 1990; Sperlagh and Vizi, 1992; Haskó and Szabó, 1998), and occupies adenosine A1, A2A, A2B and A3 receptors on target cells. Adenosine or selective agonists of the different adenosine receptors are important regulators of the production of inflammatory mediators in endotoxemia (Rose et al., 1988; Schrier et al., 1990; Le Vraux et al., 1993; Parmely et al., 1993; Bouma et al., 1994; Cronstein, 1994). Recently, a role for the adenosine A3 receptor in the modulation of immune response has been proposed, since the suppression of tumor necrosis factor-α production (Le Vraux et al., 1993; Haskó et al., 1996), NO formation (Haskó et al., 1996; Moochhala et al., 1996), or the inhibition of major histocompatibility complex-unrestricted cytolytic activity of natural killer cells (Hoskin et al., 1994) by selective A1 and A2 receptor agonists was not clearly characteristic of either the A1 or A2 subtype. Moreover, in recent studies, using selective agonists of adenosine A3 receptors, we and others have demonstrated that the stimulation of this receptor subtype potently inhibits tumor necrosis factor-α (Haskó et al., 1996; McWhinney et al., 1996; Sajjadi et al., 1996) and augments interleukin-10 (Haskó et al., 1996) production.

The aim of the present study was to further characterize the effect of adenosine A3 receptor activation on the course of inflammatory processes. Using N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA), a selective agonist of adenosine A3 receptors (Gallo-Rodriguez et al., 1994), we investigated whether the stimulation of this receptor subtype modulates interleukin-12, interferon-γ, interleukin-6, interleukin-1α, corticosterone and NO production. Since up-regulation of interleukin-10 production by certain pharmacological agents (Kambayashi et al., 1995; Le Moine et al., 1996; Van der Poll et al., 1996) has been attributed to the suppression of pro-inflammatory cytokines, we have compared the effect of IB-MECA in normal and interleukin-10 deficient endotoxemic mice. Furthermore, we determined whether the activation of adenosine A3 receptors with IB-MECA prevents lipopolysaccharide-elicited lethality.

Section snippets

Animals

Male BALB/c mice (20–25 g) were purchased from Charles River Laboratories (Budapest, Hungary). Male C57BL/6 interleukin-10+/+ and C57BL/6 interleukin-100/0 mice (7-week old) were obtained from the Jackson Laboratory (Bay Harbor, ME, USA). Animals received food and water ad libitum, and lighting was maintained on a 12-h cycle. The animal experiments were performed with the approval of the Institutional Animal Care and Use Committee.

Materials

IB-MECA was purchased from Research Biochemicals International

Effect of IB-MECA on lipopolysaccharide-induced plasma interleukin-12 (p40 and p70), interferon-γ, interleukin-6, interleukin-1α, nitrite/nitrate, and corticosterone concentrations in BALB/c mice

Intraperitoneal injection of lipopolysaccharide (60 mg/kg) to BALB/c mice induced the appearance of plasma interleukin-12 p70 levels, which reproducibly peaked at 4 h (in the pg/ml range). As shown in Fig. 1a, i.p. pre-treatment of the mice with either 0.2 or 0.5 mg/kg IB-MECA 30 min before lipopolysaccharide abolished the plasma interleukin-12 p70 response, as determined at 4 h post-lipopolysaccharide. Previous studies demonstrated that the production of the p40 subunit exceeds the production

Discussion

Recently, there has been a growing interest in determining the role of adenosine A3 receptors in the modulation of immune/inflammatory processes. In a recent study using specific adenosine A1, A2 and A3 receptor agonists and antagonists, it was demonstrated that inhibition of tumor necrosis factor-α production by lipopolysaccharide-stimulated U937 (human monocyte) cells was mainly an adenosine A3 receptor-mediated process (Sajjadi et al., 1996). Similarly, adenosine receptor agonists, in a

Acknowledgements

The expert technical assistance of Mr. P. Hake, Mr. M. O'Connor, Mrs. E. Tóth and Miss J. Benkô, is gratefully acknowledged. This work was supported by a grant from the Hungarian Medical Research Council to G.H. (ETT 200/96).

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