Inhibition of microglial inflammatory responses by norepinephrine: effects on nitric oxide and interleukin-1β production
© Dello Russo et al; licensee BioMed Central Ltd. 2004
Received: 18 March 2004
Accepted: 30 June 2004
Published: 30 June 2004
Under pathological conditions, microglia produce proinflammatory mediators which contribute to neurologic damage, and whose levels can be modulated by endogenous factors including neurotransmitters such as norepinephrine (NE). We investigated the ability of NE to suppress microglial activation, in particular its effects on induction and activity of the inducible form of nitric oxide synthase (NOS2) and the possible role that IL-1β plays in that response.
Rat cortical microglia were stimulated with bacterial lipopolysaccharide (LPS) to induce NOS2 expression (assessed by nitrite and nitrate accumulation, NO production, and NOS2 mRNA levels) and IL-1β release (assessed by ELISA). Effects of NE were examined by co-incubating cells with different concentrations of NE, adrenergic receptor agonists and antagonists, cAMP analogs, and protein kinase (PK) A and adenylate cyclase (AC) inhibitors. Effects on the NFκB:IκB pathway were examined by using selective a NFκB inhibitor and measuring IκBα protein levels by western blots. A role for IL-1β in NOS2 induction was tested by examining effects of caspase-1 inhibitors and using caspase-1 deficient cells.
LPS caused a time-dependent increase in NOS2 mRNA levels and NO production; which was blocked by a selective NFκB inhibitor. NE dose-dependently reduced NOS2 expression and NO generation, via activation of β2-adrenergic receptors (β2-ARs), and reduced loss of inhibitory IkBα protein. NE effects were replicated by dibutyryl-cyclic AMP. However, co-incubation with either PKA or AC inhibitors did not reverse suppressive effects of NE, but instead reduced nitrite production. A role for IL-1β was suggested since NE potently blocked microglial IL-1β production. However, incubation with a caspase-1 inhibitor, which reduced IL-1β levels, had no effect on NO production; incubation with IL-receptor antagonist had biphasic effects on nitrite production; and NE inhibited nitrite production in caspase-1 deficient microglia.
NE reduces microglial NOS2 expression and IL-1β production, however IL-1β does not play a critical role in NOS2 induction nor in mediating NE suppressive effects. Changes in magnitude or kinetics of cAMP may modulate NOS2 induction as well as suppression by NE. These results suggest that dysregulation of the central cathecolaminergic system may contribute to detrimental inflammatory responses and brain damage in neurological disease or trauma.
KeywordsNitric Oxide Noradrenaline Interleukin-1β Cytokines Caspase cAMP
Microglial activation including the production of pro-inflammatory cytokines and reactive oxygen species is now recognized as a key component of several neurological diseases including Multiple Sclerosis (MS) and Alzheimer's Disease (AD); as well as other conditions in which trauma, infection, or injury leads to inflammatory activation. Activated microglia produce the free radical NO synthesized by the inducible form of the enzyme nitric oxide synthase (iNOS or NOS2). NOS2 can be induced in enriched cultures of microglial cells upon treatment with proinflammatory cytokines or bacterial endotoxin [1–3], as well as in rodent brains following peripheral or intraparenchymal introduction of inflammatory inducers . In some cases NOS2 expression was dependent upon IL1β production , and some anti-inflammatory treatments were shown to reduce both microglial IL-1β as well as NOS2 expression ( for review). However other studies reported distinct, and in some cases opposite effects of anti-inflammatory treatments upon IL-1β versus NOS2 expression . Thus, the precise role for IL-1β in regulating NOS2 expression in microglia requires further study.
We demonstrated that the neurotransmitter norepinephrine (NE) prevents induction of NOS2 in rat cortical astrocytes [7, 8], and more recently in vivo that depletion of NE exacerbates the cortical inflammatory response to amyloid beta (Aβ) . Similarly, others have shown that NE reduces astroglial expression of pro-inflammatory cytokines including IL1β and TNFα [10–13] and of cell adhesion molecules . The effects of NE appear to involve activation of β-adrenergic receptors (β-ARs) and elevation of intracellular cAMP, and in most cases lead to suppression of astrocytic inflammatory responses . Perturbation in NE levels, or dysfunction in NE signaling might therefore exacerbate inflammatory responses and thus contribute to neurological damage, for example in AD and Parkinson's disease where noradrenergic locus coeruleus (LC) neurons are lost [16, 17], or in MS where astrocytic β-AR expression is reduced [18, 19].
Rat cortical microglia express all different types of ARs , and treatment with NE results in increased levels of cAMP within the cells which can be inhibited by the β-AR non selective antagonist propanol . However the cellular effects of NE on microglial inflammatory responses are less well characterized. NE reduced NO production in N9 microglial cells  and in rat microglia , but increased IL-1β mRNA in rat microglia . Other agents which increase microglial cAMP (analogs such as dibutyryl-cyclic AMP (dbcAMP), activators of adenylate cyclase (AC), or PGE2) also modulate inflammatory responses, however in contrast to astrocytes, both up as well as down regulation of NOS2 and IL-1β has been observed [23, 24]. Since the regulation of microglial NOS2 differs from astroglial NOS2  it is not surprising that anti-inflammatory treatments which attenuate astrocyte NOS2 or IL-1β may have distinct actions in microglial cells.
To better understand how NE reduces microglial inflammatory responses, we examined the effects of NE on NOS2 expression in rat cortical microglial cells. We observed that, as found for astrocytes, NE dose-dependently blocked microglial NOS2 expression, via β2-ARs activation. In the same cells, NE more potently reduced IL-1β production, reaching close to 100% attenuation at low concentrations of NE (1 to 10 μM). However, additional experiments suggest that while NE inhibits both these factors, the suppression of NOS2 expression is not directly due to the reduction of IL-1β levels. These findings indicate that, at least in vitro, microglial NOS2 expression is not dependent upon IL-1β production and therefore suggest that anti-inflammatory treatments designed to reduce IL-1β may be without effect on NOS2 levels.
Cell culture reagents (DMEM, and antibiotics) were from Cellgro Mediatech. Fetal calf serum (FCS) and DMEM-F12 were from GIBCO Life Technologies. Lipopolysaccharide (LPS, Salmonella typhimurium), NE, the NOS2 inhibitor (2-amino-dihydro-6-methyl-4H-1,3-thiazine, AMT) and the peptide aldehyde inhibitor benzyloxycarbonyl-Ile-Glu (Ot2butyl) 2Ala-leucinal (ZIE) were from Sigma. Adrenergic agonists and antagonists were from BIOMOL Research Laboratories. The protein kinase (PK) A inhibitors (KT-5270 and H-89) and activators (dbcAMP), the AC inhibitors (SQ 22536 and MDL-12,330A), the interleukin receptor antagonist (IL-1ra) and the irreversible cell permeable caspase-1 inhibitor (Ac-YVAD-CMK) were from Calbiochem (San Diego, CA). Taq polymerase and cDNA synthesis reagents were from Promega. Anti IkBa (SC-371) rabbit polyclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). NOD.ICE-/- mice, which lack functional caspase-1, and therefore do not produce mature IL-1β or IL-18  were obtained from Jackson Laboratories.
Experiments were carried out the day after the isolation from the astrocyte monolayer in DMEM-F12. In preliminary experiments, we assessed cell viability in presence of different concentrations of FCS, measuring lactic dehydrogenase (LDH) release in the incubation medium as an index of cell toxicity. We found a significant increase in LDH activity if the cells were incubated in serum free medium (see also ) or in medium containing 1% FCS; therefore all experiments were carried out in 10% FCS.
The levels of IL-1β in the incubation medium were detected by specific ELISA assays. For rat IL-1β we used an ELISA kit purchased by R&D System Inc and performed according to the manufacturer's instructions. For the assessment of IL-1β released by ICE-deficient microglial cells, we used an ELISA specific for mouse IL-1β (BD OptEIA™ Set, BD Bioscience).
NOS2 induction and activity measurements
NOS2 was induced in microglial cells by incubation with bacterial endotoxin LPS. NOS2 induction was assessed indirectly by nitrite production in the cell culture media . An aliquot of the cell culture media (80 μl) was mixed with 40 μl of Griess reagent and the absorbance measured at 550 nm. In preliminary studies, we found that the LPS dependent nitrite production was greater when cells were incubated in DMEM-F12 medium, as compared to DMEM alone; and therefore all studies were carried out in DMEM-F12.
In some experiments, we assessed total levels of nitrites in the incubation media after enzymatic reduction of nitrates to nitrites . Briefly, samples were incubated with nitrate reductase purified from Aspergillus (EC 18.104.22.168), reduced β–NADPH, and FAD for 2 hr at 37°C to convert nitrates into nitrites. Excess β–NADPH was removed by incubating the samples for 30 min at 37°C in presence of LDH from rabbit muscle (EC 22.214.171.124) and pyruvate (all reagents from Sigma). Samples were assayed before and after nitrate reduction by the Griess method, to obtain nitrite levels and calculate the ratio of nitrites/nitrates. The nitrite concentration was calculated from a NaNO2 standard curve, and complete conversion of nitrate into nitrite was confirmed by including a standard curve of NaNO3 in each test.
NOS2-derived NO production was measured by the oxidation of the cell-permeable fluorogenic probe, 2',7-dichlorodihydrofluorescein diacetate (H2DCF-DA) . Once inside the cells, H2DCF-DA is deacetylated by cytosolic esterases to free H2DCF, which can be oxidized to the fluorescent compound dichlorofluorescein (DCF). This reaction is catalyzed in vitro by the formation of the nitrogen radical peroxinitrite, while hydrogen peroxide and superoxide were found ineffective by themselves . Since peroxinitrite is formed by reaction of NO with superoxide, we used oxidation of H2DCF as a marker of NO production. Briefly, cells were activated by LPS for different periods of time. At the end of each experiment the incubation media was replaced by balanced salt solution (BSS, 124 mM NaCl, 5.8 mM KCl, 10 mM dextrose, 20 mM Hepes, 0.3 mM CaCl2(H2O)2) . Cells were incubated in plain BSS or in BSS containing 100 μM AMT, to selectively block NOS2 activity, for 20 min. At the end of this pre-incubation period 20 μM H2DCF-DA was added to the cells, which were incubated for 45 min at 37°C in the incubator. The fluorescence signal due to H2DCF oxidation within the cells was quantified using a plate fluorescence reader (GENios Multi-Detection Reader, TECAN) using 485 nm as excitation and 535 nm as emission wavelength.
Total cytoplasmic RNA was prepared from cells using TRIZOL reagent (Invitrogen); aliquots were converted to cDNA using random hexamer primers, and mRNA levels estimated by RT-PCR. The primers used for NOS2 detection were 1704F (5' CTG CAT GGA ACA GTA TAA GGC AAA C-3'), corresponding to bases 1704–1728; and 1933R (5' CAG ACA GTT TCT GGT CGA TGT CAT GA-3'), complementary to bases 1908–1933 of the rat NOS2 cDNA sequence which yield a 230 bp product. The primers used for glyceraldehyde 3- phosphate dehydrogenase (GDH) detection were 796F (5'-GCC AAG TAT GAT GAC ATC AAG AAG) and 1059R (5' TCC AGG GGT TTC TTA CTC CTT GGA) which yield a 264 bp product . Quantitative changes in mRNA levels were estimated by real time PCR using the following cycling conditions: 35 cycles of denaturation at 94°C for 10 s; annealing at 61°C for 15 s; and extension at 72°C for 20 s; followed by 2 min at 72°C, in the presence of SYBR Green (1:10,000 dilution of stock solution from Molecular Probes) carried out in a 20 μL reaction in a Corbett Rotor-Gene (Corbett Research) . Relative mRNA concentrations were calculated from the take-off point of reactions using the software included in the unit. At the end of real time PCR, the products were separated by electrophoresis through 2% agarose gels containing 0.1 μg/ml ethidium bromide to ensure production of correct sized product.
After desired incubations, cells were lysed using 8 M urea. The protein content in each sample was determined by Bradford's method using bovine serum albumin as standard. Ten μg of proteins were mixed 1: 3 with 3x gel sample buffer (150 mM Tris-HCl pH 6.8, 7.5% SDS, 45% glycerol, 7.5% of bromophenol blue, 15% β-mercaptoethanol), boiled for 5 min and separated through 10% polyacrylamide SDS gels. Apparent molecular weights were estimated by comparison to colored molecular weight markers (Sigma). After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes by semi-dry electrophoretic transfer. The membranes were blocked with 10% (w/v) low-fat milk in TBST (10 mM Tris, 150 mM NaCl, 0.1% (w/v) Tween-20, pH 7.6) for 1 h, and incubated in the presence of anti-IκBα antibody (at 1:1,500 dilution) overnight with gentle shaking at 4°C. The primary antibody was removed, membranes washed 4 times in TBST, and further incubated for 1 h at room temperature in the presence of anti-rabbit IgG-HRP secondary antibody, diluted 1: 7,000. Following 4 washes in TBST, bands were visualized by incubation in enhanced chemiluminescence reagents for 1 min and exposure to X-ray film for 5 min.
All experiments were done at least in triplicate. Data are analyzed by one or two way ANOVA followed by Dunnett's multiple comparison or Bonferroni post hoc tests and P values < 0.05 were considered significant.
LPS induces NOS2 expression in microglia
NE inhibits nitrite accumulation and NO production
NE effects are mediated by β2-ARs and may involve cAMP
NE reduces NOS2 mRNA and increases IkBα levels
Quantitative RT-PCR analysis (Figure 8) showed that LPS increased NOS2 mRNA steady state levels approximately 15-fold versus control values after 4 hr of incubation, and further increased levels (to roughly 50-fold control levels) at 24 hr incubation. The presence of NE reduced the increase in NOS2 mRNA levels at both 4 and 24 hr, suggesting an effect of NE at the transcriptional and or post-transcriptional level.
NE reduces IL-1β release
Effects of blocking IL-1β production on NOS2 expression
Consistent with previous reports, in the present study we show that rat cortical microglia can be activated in vitro by low doses of LPS leading to NOS2 expression, NO production and nitrite accumulation. Under our experimental conditions, co-incubation with NE (0.1–10 μM) inhibited LPS-dependent NOS2 expression and NO and nitrite production, via activation of β2-ARs most likely mediated by elevation of intracellular cAMP. Although NE has a high affinity for, and at low doses (100 nM to 1 μM) can increase cAMP via microglial β1 and β3 ARs, even greater increases in cAMP were found at higher (1–10 μM) NE concentrations which are needed to activate β2-ARs . Thus, the amounts of NE needed to reduce NOS2 expression may reflect a requirement to activate β2-ARs in our studies, although other non-receptor mediated effects cannot be ruled out. Previous studies of adrenergic regulation of microglial NOS2 are limited: isoproterenol decreased NO release ; and in one study , NE, terbutaline (a β2-AR agonist), dobutamine (a β1-AR agonist) as well as phenylephrine (an α1-AR agonist) all reduced NO production despite different effects on cAMP elevation; suggesting that adrenergic stimulation can attenuate NOS2 irrespective of effects on cAMP.
Our results are consistent with several reports showing that intracellular levels of cAMP modulate microglial NOS2 expression. NOS2 expression was reduced by cAMP analogs in microglia ; by PGE2 (as well as FSK and dbcAMP) in enriched microglia [43, 44]; and in mixed neuron:microglial co-cultures . Microglial NOS2 was also reduced by treatment with phosphodiesterase (PDE) inhibitors [38, 42–45]; as well as other agents which increase cAMP, including melanocortin peptides , and conditioned media from T. gondii infected astrocytes . However, NOS2 is not always suppressed by elevated cAMP, and there are several studies showing that in contrast to being inhibitory, cAMP potentiates NOS2 expression . For example, dbcAMP or IBMX treatment increased microglial NOS2 expression and activity due to Aβ . The potentiating effects of cAMP appear to be mediated through activation of C/EBP family proteins which can be stimulatory , rather than through activation of CREB proteins which may be inhibitory ; and in macrophages may include activation of other kinases including PKC isoforms and p38 MAPK . Hence, activation of distinct cAMP-dependent transcription factors could account for observation of both activation as well as suppression by cAMP in microglial cells.
It should be pointed out that studies using dbcAMP should be interpreted cautiously since dbcAMP must first be metabolized to its active form, monobutyryl cAMP, a reaction catalyzed by intracellular esterases as well as extracellularly in the presence of serum, and that also releases the butyryl group from the 5'-position. The anti-inflammatory effects of dbcAMP on NO production could therefore be due, in part, to production of sodium butyrate which in rat primary microglial cells can reduce NO production and IL6 and TNFα release . Nevertheless, findings that the effects of NE are mediated via β2-ARs which primarily increase intracellular cAMP, and also are mimicked using the βAR agonist isoproterenol are consistent with the idea that NE actions involve increases in cAMP.
Although PKA is a primary target for activation by cAMP, we found that selective PKA inhibitors did not reverse the inhibitory effects of NE, suggesting that other cAMP-dependent signaling pathways, such as the newly characterized EPAC/RAP system  may mediate NE inhibitory actions in microglia. However interpretation of results with PKA inhibitors are complicated by the fact that these inhibitors blocked LPS-dependent nitrite production (Figure 6B), suggesting a role for PKA activation in NOS2 induction.
Similarly, our results show that the AC inhibitor SQ22536 did not reverse NE actions, and by itself reduced LPS-induced NOS2 activity and expression (Figure 7). This finding is in contrast with previous studies showing no effects of this agent (or other AC inhibitors) on NOS2 induction, yet able to reverse NOS2 suppression due to activation of the prostaglandin EP2 receptors by PGE2 . However, in the same study contradictory effects of AC activation on NOS2 were observed, since sulprostone, a potent agonist for the EP1 and EP3 receptors which inhibits AC activity, inhibited LPS-induced nitrite production; furthermore nitrite production was also increased at the higher concentrations of isoproterenol (>100 nM), the AC activator forskolin (FSK, > 100 μM) and dbcAMP (>10 μM). AC activators also had contrasting effects on Aβ induced nitrite production in microglia, where low doses of forskolin (10 to 50 μM) increased NO release, and a higher dose (100 μM) reduced NO release . An understanding of the contrasting effects of PKA and AC inhibitors on nitrite production and NOS2 expression may therefore help to explain reported divergent effects of cAMP on microglial NOS2.
The effects of increasing cAMP levels on microglial IL-1β production and expression are also conflicting. Thus, Si et al.  showed that the PDE inhibitor propentofylline reduced LPS induced TNFα and IL-1β release; Caggiano and Kraig [12, 51] showed that PGE2 acting via EP2 receptors (and increased cAMP) reduced IL-1β production; and Cho et al.  showed that the dopamine metabolite NAMDA which increases cAMP and CREB activation reduced IL-1β mRNA levels. In contrast, Hetier  found that the β-AR agonist isoproterenol reduced LPS induced IL-1β as well as TNFα production, however while TNFα mRNA was reduced, IL-1β mRNA was increased. Tomozawa et al.  similarly found that isoproterenol (and dbcAMP) increased IL-1β mRNA in microglial (although not in astrocytes); and Petrova et al.  reported that PGE2 also reduced IL-1β secretion, but increased IL-1β mRNA levels. More recently, Woo et al  showed that dbcAMP reduced TNFα expression, but increased IL-1β expression in BV2 cells; and Tanaka et al.  showed in that various β1- and β2-AR agonists alone could increase IL-1β mRNA levels in rat microglia. Our data is therefore the first to demonstrate the effects of an endogenous neurotransmitter on NOS2 expression and IL-1β levels in stimulated microglial cells.
The suppression by NE of IL-1β production was similar to that seen for NOS2, which suggested that the ability of NE to reduce NOS2 may be related to its ability to reduce IL-1β. However, several features suggest that these may be independent events. Thus, in contrast to suppression of NOS2, the effects of NE on IL-1β were observed at concentrations lower than that needed for maximal inhibition of NOS2 expression, and resulted in greater extent of inhibition (over 80% inhibition of IL-1β versus 30% of NO or nitrite production). Furthermore, NE was able to reduce nitrite production in caspase-1 deficient cells (Figure 10C) demonstrating that effects of NE on IL-1β are not necessary to observe effects on NOS2. Several previous reports suggest distinct regulation of microglial NOS2 and IL-1β. Thus, microglial cells cultured in the presence of astrocytes lost their ability to produce NOS2 in response to LPS, although their IL-1β release was unaffected . Petrova et al.  and Si et al.  showed cAMP dependent reductions in IL-1β production with no effect on NO production; and Woo et al  showed increased IL-1β expression due to dbcAMP with no effect on NO production. More recently, treatment of LPS activated microglia with malonic acid C60 derivatives reduced NOS2 mRNA expression, although these same reagents increased the release of IL-1β . From these studies, it is clear that there is no necessary concordance between the regulation of IL-1β expression (or production) and that of NOS2 expression (or activity).
In general, the role that IL-1β plays in inducing glial (astrocytes or microglial) NOS2 is not clear. In astrocytes, IL-1β in combination with other cytokines (IFNγ and/or TNFα) can induce rodent NOS2 [25, 56], and a few reports suggest that IL-1β alone may induce rodent astrocyte NOS2 . In contrast, in human fetal and adult astrocytes, IL-1β alone can induce NOS2 [58, 59] which is greatly increased by other cytokines . Although astrocyte NOS2 induction can, in some cases, be reduced by treatment with IL-ra (hypoxia, ; using CM obtained from Gp41 activated of microglia, ; using Aβ stimulation, ) it is likely that other factor(s) are released which contribute to NOS2 induction. In contrast to astrocytes, there are no clear reports to indicate that IL-1β alone will induce microglial NOS2, and in fact human microglial appear more refractory to NOS2 inducers than do rodent cells . Our results are consistent with the conclusion that the LPS induced IL-1β does not play an important role in mediating microglial NOS2 expression.
The molecular mechanism(s) by which NE reduces microglial NOS2 expression and IL-1β production and expression are not yet known. Work from several laboratories has shown in glial cells that LPS rapidly activates PK cascades which lead to phosphorylation of inhibitory IκB proteins, their degradation by the 26S proteasome, and subsequent activation of NFκB , necessary for the expression of pro-inflammatory genes . We observed that LPS induced rapid loss of the microglial IκBα protein, while co-incubation with NE reduced that loss and moreover increased IκBα levels after longer times. Several reports suggest that increases in cAMP are associated with increased levels of IκBα [66–68], and we demonstrated that NE directly increases transcription of the IκBα gene in astrocytes and reduces the overall magnitude of IkBa degradation . Taken together these data suggest that the inhibitory effects of NE in microglia may also be mediated in part by interference with the NFkB:IkB signaling pathway.
The in vivo relevance of noradrenergic regulation of inflammatory gene expression, is suggested by observations that the noradrenergic neurons of the LC are damaged or lost in AD , leading to a loss (or at least a transient loss) in noradrenergic signaling within projection areas. A possible perturbation in noradrenergic signaling is also implicated in MS, since it has been shown that treatment with β-AR agonists [70, 71] can provide protection in animal models of MS, and more recently that the levels of β2-ARs in astrocytes are decreased in MS patients as compared to healthy controls . We recently demonstrated that LC loss increases both the magnitude as well as the duration of the inflammatory responses in vivo including the extent of microglia activation . Furthermore, we showed that central NE depletion led to a dramatic decrease in cortical levels of the IkBa protein, consistent with the idea that NE normally keeps the IkBa gene transcriptionally active . Together with the knowledge that IL-1β plays a prominent role in the pathogenesis of neurodegenerative diseases, such as AD , and in view of the fact that microglial NOS2 expression is implicated in the damage occurring in MS [75, 76], AD  and cerebral ischemia [78, 79], it may be of value to consider possible therapeutic strategies to increase microglial β-AR activation or intracellular cAMP levels.
List of abbreviations
inducible nitric oxide synthase
interleukin 1-receptor antagonist
fetal calf serum
benzyloxycarbonyl-Ile-Glu (Ot2butyl) 2Ala-leucinal
We thank Anthony Sharp for assistance with cell culture studies, and Dr. Pilar Mercado for help with ELISA measurements. This work was supported in part by NIH grants NS31556 and NS044945 to DLF and a postdoctoral award from the National Multiple Sclerosis Society (CDR).
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