Background
Microglia, the innate immune cells of the brain, constantly screen their microenvironment and transform into an "activated" state in response to brain lesions, e.g., toxic lesions or debris and degenerating neurons [
1,
2] (for review see [
3]). Once activated, microglia secrete pro- and anti-inflammatory mediators such as cytokines and prostaglandins (for review see [
4]).
In vitro stimulation with lipopolysaccharide (LPS), the endotoxin of gram-negative bacteria, results in the secretion of neurotoxic and pro-inflammatory mediators. LPS triggers the activation of microglial cells via the anchored surface myeloid glycoprotein CD14 [
5]. CD14 has also been found to bind to amyloid-β (Aβ), the major compound found in amyloid plaques in the brains of patients with Alzheimer's disease (AD) [
6].
LPS is known to induce the production of cyclooxygenase-2 (COX-2) enzyme in microglial cells
in vitro [
7‐
9]. COX-2 converts arachidonic acid released from membrane phospholipids to prostaglandin (PG) H
2. PGH
2 is then isomerized to PGE
2 by terminal prostaglandin E synthases. COX-2 has emerged as a major player in inflammatory reactions in the brain and increased COX-2 expression has been considered to contribute to neurodegeneration [
10,
11]. Elevated COX-2 expression has been described in AD [
12‐
17] and COX-2 protein content in the hippocampus of AD patients may correlate with the severity of dementia [
18] On the other hand, COX-2 has been suggested to play a physiological role in the brain for being involved in neuronal plasticity and synaptic transmission [
19,
20].
In recent years it has become evident that there may exist a crosstalk between the autonomic nervous system and the immune system during inflammation [
21,
22]. The catecholamine norepinephrine (NE) is a classical neurotransmitter with suggested immunomodulatory properties. NE is released by neurons into the synaptic cleft and may exert effects on glial cells that are in close vicinity. NE binds to α- and β-adrenergic receptors, and expression of α
1-, α
2-, β
1- and β
2-adrenegic receptors have been identified on microglia [
23‐
29].
An immunosuppressive role has been suggested for NE
in vitro since it attenuates LPS-induced microglial production of tumour necrosis factor (TNF)α, interleukin (IL)-1β, IL-6 and nitric oxide
in vitro [
24,
30,
31]. NE has been shown to reduce microglia-induced neuronal cell death [
30,
32].
Although it is known that NE has immunosuppressive properties, information of NE's effect on prostaglandins is scarce. The aim of our study was to further investigate the role of NE on eicosanoid production, namely PGE2, in primary rat microglia.
Discussion
In the present study, basal COX-2 expression in non-stimulated rat primary neonatal microglia was not detectable, suggesting that either COX-2 is generated by de novo synthesis in response to applied stimuli or, alternatively, that basal levels do not reach the threshold of immunoblot detection. NE alone induced COX-2 mRNA expression but did not affect COX-2 protein synthesis. We suppose that the COX-2 mRNA induced by NE is degraded before protein synthesis is initiated. Alternatively, a second stimulus, such as LPS, is required to set off translation of COX-2.
Different kinases are important in the regulation of COX-2 in LPS-stimulated microglia [
37], and it has been demonstrated that NE increases the activity of mitogen-activated protein kinases (MAPK) [
38] as well as transcription factors [
39]. Kan et al. (1999) demonstrated that, in neonatal rat cardiac myocytes, NE alone increases the activity of MAPK and, although IL-1β alone does not induce the same effect, the co-addition of IL-1β and NE results in an enhanced MAPK activity in comparison to the substances alone [
38]. Salmeterol and isoproterenol, β2- and non-selective β-adrenergic receptor agonists, respectively, enhance the phosphorylation of p38 MAPK and extracellular signal-regulated kinases (ERK) in peritoneal macrophages and RAW264.7 [
40,
41]. Interestingly, pretreatment with isoproterenol decreases the release of TNFα, IL-12 and NO in LPS-stimulated macrophages. On the contrary, isoproterenol reduces the release of these same mediators after PMA stimulation, indicating that the effect of isoproterenol might depend on the stimulus [
42].
Recently, Morioka et al. (2009) demonstrated that, in rat spinal microglia, NE reduces phosphorylation of p38 MAPK, induced by ATP, via β
1- and β
2-receptors [
43]. The deactivation of p38 would lead to a decrease in COX-2 protein synthesis, since p38 is involved in COX-2 mRNA stabilization [
44]. As such, it is possible that the observed increase in COX-2 mRNA may be due to increased transcription or increased stability of mRNA in rat microglia by activation of certain transcription factors and/or kinases. That could explain the increase in the COX-2 mRNA with incubation of cells with NE alone, or the enhancement of COX-2 protein synthesis when associated with LPS.
On the other hand, many post-transcriptional factors contribute to the translation of mRNA, which might not be affected by NE. This could explain the lack of effect of NE on COX-2 protein synthesis. For example, it is known that LPS induces the activation of the mammalian target of rapamycin (mTOR) [
45]. Activation of mTOR induces translation of different mRNA through its downstream targets such as the ribosomal p70S6 kinase and the initiation factor 4E-binding protein 1 [
46]. Although we did not test this possibility, it seems feasible that NE
per se cannot activate the machinery responsible for the translation of COX-2 in rat microglia, but potentiates protein synthesis by increasing transcription or protein stability.
As shown before, LPS, 10 ng/ml, increases COX-2 mRNA and protein expression at 4 and 8 h, respectively [
8]. Co-stimulation with NE already at low concentrations results in earlier and a markedly enhanced induction of COX-2 mRNA and protein levels. Similar to our results, other groups have also shown that NE
per se does not increase protein synthesis, but drastically increases the effect induced by LPS. In peripheral blood monocytes and monocyte-derived macrophages, NE and epinephrine alone only show a minor effect on matrix metalloproteinase (MMP)-1 and MMP-9 production [
39]. However, a combination of the catecholamine with LPS further enhances the increased production of MMP-1 and MMP-9.
Next, we investigated whether the increased intracellular levels of COX-2 evoked by LPS plus NE causes elevated levels of PGE2. In our experiments, NE plus LPS further increased the levels of PGE2. Interestingly, after 24 h stimulation with NE plus LPS, COX-2 mRNA and protein levels are similar to the increase observed in LPS alone. However, the increase observed in PGE2 is about three-fold higher in LPS plus NE than with LPS alone. Thus, it is possible that the combination of LPS and NE might induce the expression or increase the activity of other enzymes involved in PGE2 synthesis, such as phospholipase A2 and/or PGE synthases.
Dependent on the experimental setting, PGE
2 can exert either neuroprotective or neurodetrimental effects. Agents reducing PGE
2 synthesis in
in vitro and
in vivo models of chronic neurodegenerative diseases like Parkinson's disease or AD have been demonstrated to be neuroprotective due to their anti-inflammatory effects [
47‐
52]. On the other hand, exogenous PGE
2 protects neurons from LPS-induced cell death by reduction of NO and reactive oxygen species [
53]. Direct administration of PGE
2 into the brain has also been shown to reduce microglial activation and TNF-α expression in brain parenchyma induced by intraperitoneal LPS injection [
54]. In addition, PGE
2 protects neurons in culture from different types of noxious stimuli [
29,
55,
56].
Localized inflammatory responses in the brain parenchyma have been associated with the pathogenesis and progression of AD. Inhibition of neuroinflammation has been identified as a potential therapeutic target [
57‐
60]. COX-2 expression is elevated in the AD brains [
14,
18] and PGE
2 is accumulated in the cerebrospinal fluid of AD patients [
61]. It was therefore reasonable to hypothesize that inhibition of COX-2 may have a therapeutic potential in AD. Despite convincing evidence in epidemiological studies on the prevention of AD through long-term treatment with non-steroidal anti-inflammatory drugs, most clinical trials have failed to show beneficial effects [
62‐
65], suggesting that either the molecular target or the therapeutic window has been missed.
PGE
2 acts on four different receptors, EP1-EP4 [
66], of which microglia express three, namely EP1, EP2, and EP3; the latter having been exclusively detected in activated microglia [
67]. Microglial EP2 receptors are known to enhance neurotoxic activities [
10,
50‐
52,
68]. This would suggest that enhanced secretion of PGE
2 through NE might increase microglial toxicity. However, the role of PGE
2 may be far more complex due to the presence of other receptor subtypes on microglia. So far, NE-mediated regulation of EP receptor expression on microglia has not been studied.
In this study, we could show that the observed effect of NE is mediated by β-adrenoreceptor agonists. This corresponds to the findings of Minghetti and Levi, who showed that the non-selective β-adrenergic agonist isoproterenol increases COX-2 protein and PGE
2 synthesis in microglial cells [
8]. In our experiments, both β
1- and β
2-receptors seem to mediate the enhanced effect of COX-2 production. Our data also indicate a lack of involvement of α-adrenoreceptors. The use of relatively-selective β-adrenoreceptors allowed us to further confirm the participation of β-adrenoreceptors in the enhancement of LPS-induced COX-2 expression. Based on our data, both β-adrenoreceptors subtypes seem to be involved. The antagonists used in this study, CGP20712A (β
1-antagonist) and ICI118,551 (β
2-antagonist) are widely used to discern the role of β-adrenoreceptor subtypes [
69,
70]. Terbutaline is considered a relatively selective β
2-agonist, but it also binds to the β
1-adrenoreceptor [
71]. As with any pharmacological agent, these compounds are not 100% selective, therefore future studies utilizing knockout animals will be needed to fully clarify the role of distinct β-adrenoreceptor subtypes.
We did not detect that stimulation of α-adrenoreceptors results in any significant increase in COX-2 protein levels. Of note, stimulation of β-adrenoreceptors increases levels of cAMP in microglial cells, mainly through activation of the β
2-adrenoreceptor [
25]. Raised intracellular cAMP levels are known to suppress activation of microglial cells [
54,
72,
73]. Increased PGE
2 levels may in addition stimulate the microglial EP2 receptor, which is linked to cAMP formation [
74] and thereby contribute to the inactivation of microglia. Other cell culture experiments have shown that exogenous PGE
2 results in decreased levels of pro-inflammatory cytokines such as TNF-alpha and IL-12 in LPS-stimulated microglia [
75‐
77]. In concordance with these results, increased cAMP levels via stimulation of β-adrenoreceptors inhibits the production of macrophage inflammatory protein (MIP)-1α, which is known to activate macrophages to secrete pro-inflammatory cytokines [
78].
Our data suggest that NE has a strong effect on microglial inflammatory responses, suggesting that NE is an active modulator of microglial activation. This may be important in AD, a disease in which early loss of noradrenergic locus coeruleus (LC) neurons has been observed [
79]. Depletion of LC neurons by injection of the neurotoxin DSP4 increases the levels of inflammatory mediators like iNOS, IL-1β and IL-6 [
80].
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JCMS contributed to the design of the study, carried out western blot analysis, performed the statistical analysis and wrote the manuscript; BLF directed the work of the study, reviewed the data and the manuscript; EH participated in western blot analysis and prostaglandin measurements; ECJ and ACPO provided consultation and reviewed the manuscript; MTH was involved in drafting the manuscript and revised it critically; MH conceived of the study, directed the work and reviewed the manuscript. All authors read and approved the final manuscript.