Combined anti-inflammatory effects of β₂-adrenergic agonists and PDE4 inhibitors on astrocytes by upregulation of intracellular cAMP

Abstract

Inflammation is an important hallmark of all neurodegenerative diseases and activation of different glial populations may be involved in the progression of some of these disorders. Especially, the activation of astroglia can lead to long-term detrimental morphological changes, such as scar formation. Therefore, improved strategies to modulate inflammation in these cells are currently being investigated. We investigated the interaction of phosphodiesterase (PDE) 4 inhibitors, such as rolipram, with other agents raising cellular cAMP levels. When used alone, none of the PDE4 inhibitors increased cAMP levels. The adenylate cyclase activator forskolin, the β₂-adrenergic agonist clenbuterol and the mixed β₁/β₂-adrenergic agonist isoproterenol increased intracellular cAMP levels of cortical murine astrocytes. This increase was synergistically elevated by rolipram or the PDE4 inhibitor RO-201724, but not by inhibition of PDE3. Inflammatory stimulation of the cells with the cytokines TNF-α, IL-1β and IFN-γ strongly induced PDE4B and augmented overall PDE4 activity, while PDE3 activity was low. Clenbuterol and forskolin caused downregulation of cytokines and chemokines such as IL-6 and MCP-1. This effect was further enhanced by rolipram, but not by the PDE3 inhibitor milrinone. The cAMP-raising drug combinations attenuated the upregulation of TNF-α and IL-6 mRNA and the secretion of IL-6, but did not affect initial NF-κB signalling triggered by the stimulating cytokines. These results indicate that PDE4 may be a valuable anti-inflammatory target in brain diseases, especially under conditions associated with stimulation of cAMP-augmenting astrocyte receptors as is observed by clenbuterol treatment.

Introduction

Inflammation is associated with many disorders affecting the central nervous system (CNS) (Saijo et al., 2010), and some studies suggest that anti-inflammatory treatment may slow disease progressions (Hartmann et al., 2003, Villoslada et al., 2008, Fletcher et al., 2010). Although they are not regarded as traditional immune cells, astrocytes are capable of mounting a pronounced inflammatory response when they are triggered by endogenous inflammatory mediators, or by ligands of certain toll-like receptors (Chung and Benveniste, 1990, Choi et al., 1999, Falsig et al., 2004, Bsibsi et al., 2002). The spectrum of factors secreted and upregulated after stimulation with a cytokine mix is remarkably similar to that of activated microglia and astrocytes are considered a second important line of host defence for the CNS (Minagar et al., 2002, Falsig et al., 2006, Falsig et al., 2008).

Several studies have shown the regulation of glial inflammatory mediators by noradrenaline (Feinstein et al., 2002, Dello et al., 2004, Simonini et al., 2010). Some of these effects may be related to the increase in intracellular cAMP levels caused by the selective stimulation of β-adrenoceptors (Szabo et al., 1997). Accordingly, specific β-adrenergic agonists have been shown to modulate inflammation by inhibiting TNF-α production and by up-regulating IL-10 expression (Szabo et al., 1997, Farmer and Pugin, 2000). Especially the agonist clenbuterol appears interesting as it shows neuroprotective effects in a mouse and rat model of cerebral ischemia, and closer investigation has shown that it not only blocks astrocytosis in this model, but also reduces the upregulation of the inflammatory marker GFAP in cultured astrocytes (Culmsee et al., 1999, Culmsee et al., 2007). The role of intracellular cAMP levels in inflammation is further supported by multiple studies showing that inhibition of phosphodiesterase (PDE) 3 and 4 is immunomodulatory (Schudt et al., 1999, Mendes et al., 2009, Pages et al., 2009). For instance, rolipram, a widely used inhibitor of PDE4, reduces inflammation and has been shown to suppress the clinical manifestations of an animal model of multiple sclerosis (Sommer et al., 1997, Block et al., 2004, de Visser et al., 2008). However, only little information is available on the effects of PDE4/PDE3 inhibition on astrogliosis in disease models.

The most prominently expressed PDE subtypes in immune cells are PDE3 and PDE4 (Lugnier, 2006). In some cell types a combined inhibition of both enzyme activities is required to obtain maximal increases of cAMP and attenuation of inflammation (Gantner et al., 1997, Gantner et al., 1999). In other cells PDE inhibitors have no effect unless they are combined with a cAMP-increasing stimulus (Selige et al., 2010a). For instance, β-adrenergic agonists and PDE inhibitors may show synergy (Bruss et al., 2008).

More information is required on the PDE spectrum of resting and activated murine astrocytes, and on how inhibition of PDE3 or PDE4 would affect the anti-inflammatory activity of β₂-adrenergic agonists. Here, we examined these questions by using the adenylate cyclase activator forskolin, the β₂-adrenergic agonist clenbuterol and the mixed β₁/β₂-adrenergic agonist isoproterenol together with specific PDE3 and PDE4 inhibitors. In murine primary cortical astrocytes, stimulated with a complete cytokine mix consisting of TNF-α, IL-1β and IFNγ, we examined how the PDE4 inhibitor rolipram, in combination with other drugs, would affect mRNA levels and secretion of mediators such as IL-6.