In this study, we examined (a) the change in MOR expression in U87 MG human astrocytes exposed to IL-1β; (b) the ability of IL-1β to up-regulate the MOR desensitized by chronic morphine exposure; and (c) the role of the IL-1 receptor in IL-1β-mediated up-regulation of the MOR. Prior work in our laboratory showed that there is an alteration in LPS-induced cytokine expression when an animal is in a morphine tolerant state [14, 30, 31].
U87 MG cells are known to possess IL-1R1, the functional receptor for the IL-1 pro-inflammatory cytokine family ; however, the opioid receptors in this cell line have not yet been characterized. Using real time RT-PCR, we first determined the basal levels of the opioid receptors in the U87 MG cells. Our results showed that U87 MG cells exhibit moderate basal expression of all three receptors: MOR, DOR and KOR. We then verified that MOR is functionally active in the U87MG cells. MOR is a G-protein coupled receptor (GPCR) and inhibits adenylyl cyclase, which in turn decreases intracellular cAMP levels . As a positive control, forskolin, which is known to activate adenylyl cyclase and increase cAMP levels, was used . We also used morphine and other MOR agonists, such as endomorphin 1 and 2, as well as a MOR antagonist, naloxone. Our results show that the morphine and endomorphins were able to decrease the cAMP levels, whereas naloxone reverses the inhibitory effect, as we had expected. This proved that the MOR is functionally active in U87 MG cells and, therefore, is a good cell model to study the MOR signaling and activity.
We then examined IL-1β’s ability to modulate the expression of the opioid receptors. IL-1β induced a significant increase in MOR, DOR, and KOR, which is consistent with prior reports that IL-1 can induce MOR expression in human neural microvascular endothelial cells . Although all three types of opioid receptors were significantly up-regulated by IL-1β, the increase in MOR was much more pronounced than DOR or KOR, suggesting that the MOR is a more potent mediator of an immune-opioid relationship than the DOR or KOR. Mohan et al. (2010) reported a similar observation in SK-N-SH neuroblastoma cells where MOR expression was increased after treatment with IL-1β. Since IL-1 cytokines are known to increase transcription through several different pathways, such as mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB), this could, in part, explain the increase in the opioid receptor transcription that is observed after IL-1β treatment [35, 36].
The ability of morphine to desensitize opioid receptors is well established, as is the ability of IL-1β to up-regulate the MOR in neural microvascular endothelial cells, neuronal cells and glial cells [3, 17, 29]. In this study, we examined the ability of IL-1β to affect desensitized MOR in U87 MG cells. A time course revealed that MOR expression increased significantly after 3 h of morphine exposure, followed by a desensitization of the MOR. After 24 h of pretreatment with morphine, there was an increase in IL-1β-induced MOR expression in U87 MG as compared to U87 MG cells not pretreated with morphine. This was potentially due to a rebound effect which occurred when the morphine-containing medium was aspirated and replaced with non-morphine containing medium. The removal of the morphine-enriched medium could have precipitated cellular morphine withdrawal, which can cause MOR up-regulation . However, morphine-desensitized U87 MG cells treated with IL-1β showed a significant increase in the MOR as compared to morphine treated cells not exposed to IL-1β. These data indicate that pro-inflammatory cytokines are able to modulate opioid receptors, both in an untreated and a morphine desensitized state in astrocytes. Pro-inflammatory cytokines, such as IL-1β, can affect the opioid-dependent pathways by stimulating the expression of the opioid receptors. Conversely, opioids, such as morphine, can induce the production of inflammatory cytokines. In rodent models, IL-1β expression was increased after administration of chronic morphine . In another study, morphine was found to activate the toll-like receptor 4 (TLR4) in addition to the MOR. TLR4 is present on immune cells, such as microglia, which, upon activation, release pro-inflammatory cytokines, such as IL-1 . Similar observations were reported in endothelial and neuroblastoma cells where morphine and IL-1β co-treatment increased MOR expression, which further substantiated our data [15, 35].
IL-1RAP, an antagonist to the IL-1 receptor, was then used to determine the role of the IL-1β receptor in the modulation of the MOR. U87 MG cells treated with a high ratio of IL-1β to IL-1RAP (1:20) showed a significant decrease in MOR expression, whereas U87 MG cells treated with a low ratio of IL-1βto IL-1RAP (1:200) showed no difference in MOR expression compared to the control (only vehicle); however, there was a significant decrease in IL-1β-induced MOR expression compared to cells treated with only IL-1β. This indicates that the effect of IL-1β on MOR expression in U87 MG cells occurs through IL-1R1. The inability of the higher concentration of IL-1RAP to completely block IL-1β-induced up-regulation of the MOR expression could be because of over-saturation of the U87 MG cells. Another reason could be that high concentrations of IL-1RAP (without co-treatment of IL-1β) can decrease basal levels of IL-1β to such an extent that there is a negative feedback mechanism. Shavit et al. reported that acute administration of IL-1RAP in mice immediately after termination of morphine-induced analgesia, resulted in an induction of analgesia suggesting the MOR was probably up-regulated and, hence, analgesia was reinstated .
Since astrocytes co-exist with neuronal cells in the nervous system, it is important to take into account the effect of one on the other. It has been seen that lipopolysaccharide (LPS) stimulates MOR expression in both neuronal and macrophage-like cell models, through accumulation of reactive oxygen species (ROS) and pro-inflammatory cytokines , leading to further immune suppression and, thus, bringing about homeostasis. This suggests that IL-1β not only stimulates MOR expression in astrocytes but also in neuronal and other immune cells, highlighting the involvement of opioid receptors in the neuroimmune axis.
Our data also simulate the conditions seen in Human Immunodeficiency Virus 1 (HIV-1) infection, where IL-1β expression is up-regulated through activation of the NLAP3 inflammasome . Since the derogatory effects of opioids on HIV-1 infection have been widely reported, it is possible that the HIV-1 induced increase in IL-1β secretion can also increase MOR expression in astrocytes, as suggested by our data.