Involvement of ERK1/2, cPLA₂ and NF-κB in microglia suppression by cannabinoid receptor agonists and antagonists

Abstract

Cannabinoids have been consistently shown to suppress microglia activation and the release of cytotoxic factors including nitric oxide, superoxide and proinflammatory cytokines. However, the underlying molecular mechanisms and whether the action of cannabinoids is coupled to the activation of cannabinoid type 1 (CB1) and type 2 (CB2) receptors are still poorly defined. In this study we observed that the CB1 and CB2 receptor non-selective or selective agonists dramatically attenuate iNOS induction and ROS generation in LPS-activated microglia. These effects are due to their reduction of phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2), cytosolic phospholipase A₂ (cPLA₂) and activation of NF-κB. Surprisingly, instead of reversing the effect of the respective CB1 and CB2 receptor agonists, the antagonists also suppress iNOS induction and ROS generation in activated microglia by similar mechanisms. Taken together, these results indicate that both cannabinoid receptor agonists and antagonists might suppress microglia activation by CB1 and CB2 receptor independent mechanisms, and provide a new insight into the mechanisms of microglia inhibition by cannabinoids.

Introduction

Microglia, the resident immune cells in the central nervous system (CNS), are thought to carry out homeostatic surveillance, screening for changes in the microenvironment [1], [2]. Upon activation, microglia undergo a dramatic transition from a resting, ramified morphology to a phagocytosis-capable, “macrophage-like” phenotype [3], [4]. Although the initial response of microglia to injury is to promote repair by removing debris and dying cells, excessive activation of microglia appears to be toxic to neighboring cells due to their release of inflammatory cytokines [5], [6], glutamate [7], [8], nitric oxide and peroxynitrite [9]. Accumulating evidence indicates that cannabinoids can modulate the function of activated microglia and the release of cytotoxic factors [10].

It has been shown that both cannabinoid receptor type 1 (CB1) and type 2 (CB2) are expressed in microglia and astrocytes [11]. Unlike the CB1 receptor, which is constitutively expressed in microglia, the expression of CB2 receptor is inducible and modulated in response to the activation stage of microglia [10], [11]. Increased expression of CB1 and CB2 receptors has been demonstrated in many inflammatory and neurodegenerative diseases, including multiple sclerosis, traumatic brain and spinal cord injury, Parkinson's disease, Huntington's disease and Alzheimer's disease [12], [13], [14], [15], [16].

Although there is a consensus that activation of these receptors can suppress the release of cytotoxic factors from activated microglia and astrocytes, it is still controversial whether and how these receptors mediate the anti-inflammatory action of cannabinoids. It has been reported that the inhibitory effect of WIN55212-2, a non-selective cannabinoid agonist, on TNF-α production from LPS-activated microglia is not altered by CB1 or CB2 receptor antagonists [17], [18]. However, other studies suggested that the release of TNFα and other proinflammatory cytokines, such as IFN-γ, IL-12 and IL-23, is attenuated by signaling pathways coupled to the cannabinoid receptor activation [19], [20]. Similarly, WIN55212-2 and other non-selective cannabinoid receptor agonists are also shown to attenuate iNOS induction in activated microglia and glutamate excitotoxicity by cannabinoid receptor dependent and independent mechanisms [21], [22], [23]. Although CB2 selective agonists have been shown to be beneficial in several animal models of neurodegenerative diseases, as well as neuropathic and inflammatory pain by inhibiting microglia activation [24], [25], [26], [27], [28], [29], [30], the selective CB1 agonists and cannabidiol (CBD), a non-CB1/CB2 receptor interactive cannabinoid, are also shown to have anti-inflammatory effects by targeting CB1 receptor and the non-CB1/CB2 receptors expressed in microglial cells, respectively [31], [32], [33], [34].

We and others have previously shown that peroxynitrite, the reaction product of nitric oxide and superoxide, is the primary toxic species released from activated microglia causing toxicity to neurons and oligodendrocytes [35], [36], [37], [38], [39]. It has been suggested that activation of cPLA₂ occurs upstream of iNOS induction in macrophages and astrocytes [40], [41], and phosphorylation of mitogen activated protein kinase (MAPK) is required for the activation of cPLA₂ [42], [43]. In this study, we examined whether the inhibitory effects of cannabinoids are due to their interference with the signaling pathways leading to iNOS induction. Using the microglia cell line BV-2 cells and primary cultures of microglia, we found that the CB1 and CB2 receptor non-selective and selective agonists attenuated iNOS induction and ROS generation in LPS-activated microglia. Surprisingly, the inhibitory effects of these cannabinoids are not reversed by the cannabinoid receptor antagonists, and, in fact, these antagonists themselves can also attenuate nitric oxide and superoxide production in activated microglia. Furthermore, the inhibitory action of both cannabinoid receptor agonists and antagonists is mediated by a similar signaling pathway, which includes inhibition of ERK1/2 and cPLA₂ phosphorylation and suppression of NF-κB activation.