Vitaliy Gavrilyuk

Noradrenergic regulation of inflammatory gene expression in brain.

It is now well accepted that inflammatory events contribute to the pathogenesis of numerous neurological disorders, including multiple sclerosis (MS), Alzheimers disease (AD), Parkinsons disease, and AIDs dementia. Whereas inflammation in the periphery is subject to rapid down regulation by increases in anti-inflammatory molecules and the presence of scavenging soluble cytokine receptors, the presence of an intact blood-brain barrier may limit a similar autoregulation from occurring in brain. Mechanisms intrinsic to the brain may provide additional immunomodulatory functions, and whose dysregulation could contribute to increased inflammation in disease. The findings that noradrenaline (NA) reduces cytokine expression in microglial, astroglial, and brain endothelial cells in vitro, and that modification of the noradrenergic signaling system occurs in some brain diseases having an inflammatory component, suggests that NA could act as an endogenous immunomodulator in brain. Furthermore, accumulating studies indicate that modification of the noradrenergic signaling system occurs in some neurodiseases. In this article, we will briefly review the evidence that NA can modulate inflammatory gene expression in vitro, summarize data supporting a similar immunomodulatory role in brain, and present recent data implicating a role for NA in attenuating the cortical inflammatory response to beta amyloid protein.

Noradrenergic depletion increases inflammatory responses in brain: effects on IκB and HSP70 expression

The inflammatory responses in many cell types are reduced by noradrenaline (NA) binding to β‐adrenergic receptors. We previously demonstrated that cortical inflammatory responses to aggregated amyloid beta (Aβ) are increased if NA levels were first depleted by lesioning locus ceruleus (LC) noradrenergic neurons, which replicates the loss of LC occurring in Alzheimers disease. To examine the molecular basis for increased responses, we used the selective neurotoxin DSP4 to lesion the LC, and then examined levels of putative anti‐inflammatory molecules. Inflammatory responses were achieved by injection of aggregated Aβ1–42 peptide and IL‐1β into frontal cortex, which induced neuronal inducible nitric oxide synthase (iNOS) and microglial IL‐1β expression. DSP4‐treatment reduced basal levels of nuclear factor kappa B (NF‐κB) inhibitory IκB proteins, and of heat shock protein (HSP)70. Inflammatory responses were prevented by co‐injection (ibuprofen or ciglitzaone) or oral administration (pioglitazone) of peroxisome proliferator‐activated receptor gamma (PPARγ) agonists. Treatment with PPARγ agonists restored IκBα, IκBβ, and HSP70 levels to values equal or above those observed in control animals, and reduced activation of cortical NF‐κB. These results suggest that noradrenergic depletion reduces levels of anti‐inflammatory molecules which normally limit cortical responses to Aβ, and that PPARγ agonists can reverse that effect. These findings suggest one mechanism by which PPARγ agonists could provide benefit in neurological diseases having an inflammatory component.

Norepinephrine Increases IκBα Expression in Astrocytes

The neurotransmitter norepinephrine (NE) can inhibit inflammatory gene expression in glial cells; however, the mechanisms involved are not clear. In primary astrocytes, NE dose-dependently increased the expression of inhibitory IκBα protein accompanied by an increase in steady state levels of IκBα mRNA. Maximal increases were observed at 30–60 min for the mRNA and at 4 h for protein, and these effects were mediated by NE binding to β-adrenergic receptors. NE activated a 1.3-kilobase IκBα promoter transfected into astrocytes or C6 glioma cells, and this activation was prevented by a β-antagonist and by protein kinase A inhibitors but not by an NFκB inhibitor. NE increased IκBα protein in both the cytosolic and the nuclear fractions, suggesting an increase in nuclear uptake of IκBα. IκBα was detected in the frontal cortex of normal adult rats, and its levels were reduced if central NE levels were depleted by lesion of the locus ceruleus. The reduction of brain IκBα levels was paralleled by increased inflammatory responses to lipopolysaccharide. These results demonstrate that IκBα expression is regulated by NE at both transcriptional and post-transcriptional levels, which could contribute to the observed anti-inflammatory properties of NE in vitro and in vivo.

Suppressive effects of ansamycins on inducible nitric oxide synthase expression and the development of experimental autoimmune encephalomyelitis

The production of nitric oxide by the inflammatory isoform of nitric oxide synthase (NOS2) in brain glial cells is thought to contribute to the causes and development of neurological diseases and trauma. We previously demonstrated that activation of a heat shock response (HSR) by hyperthermia reduced NOS2 expression in vitro, and in vivo attenuated the clinical and histological symptoms of the demyelinating disease experimental autoimmune encephalomyelitis (EAE; Heneka et al. [2001] J. Neurochem. 77:568–579). Benzoquinoid ansamycins are fungal‐derived antibiotics with tyrosine kinase inhibitory properties, and which also induce a HSR by allowing activation of HS transcription factor HSF1. We now show that two members of this class of drugs (geldanamycin and 17‐allylamino‐17‐demethoxygeldanamycin) also induce a HSR in primary rat astrocytes and rat C6 glioma cells. Both drugs dose‐dependently reduced nitrite accumulation, NOS2 steady‐state mRNA levels, and the cytokine‐dependent activation of a rat 2.2‐kB NOS2 promoter construct stably expressed in C6 cells. These inhibitory effects were partially reversed by quercetin, a bioflavonoid which prevents HSF1 binding to DNA and thus attenuates the HSR. Ansamycins increased mRNA levels of the inhibitory IκBα protein, suggesting that inhibition of NFκB activation could contribute to their suppressive effects. Finally, in C57BL/6 mice actively immunized to develop EAE, a single injection of geldanamycin at 3 days after immunization reduced disease onset by over 50%. These results indicate that ansamycins can exert potent anti‐inflammatory effects on brain glial cells which may provide therapeutic benefit in neuroinflammatory diseases.

Noradrenaline induces expression of peroxisome proliferator activated receptor gamma (PPARγ) in murine primary astrocytes and neurons

Cerebral inflammatory events play an important part in the pathogenesis of Alzheimers disease (AD). Agonists of the peroxisome proliferator‐activated receptor gamma (PPARγ), a nuclear hormone receptor that mediates anti‐inflammatory actions of non‐steroidal anti‐inflammatory drugs (NSAIDs) and thiazolidinediones, have been therefore proposed as a potential treatment of AD. Experimental evidence suggests that cortical noradrenaline (NA) depletion due to degeneration of the locus ceruleus (LC) – a pathological hallmark of AD – plays a permissive role in the development of inflammation in AD. To study a possible relationship between NA depletion and PPARγ‐mediated suppression of inflammation we investigated the influence of NA on PPARγ expression in murine primary cortical astrocytes and neurons. Incubation of astrocytes and neurons with 100 µm NA resulted in an increase of PPARγ mRNA as well as PPARγ protein levels in both cell types. These effects were blocked by the β‐adrenergic antagonist propranolol but not by the α‐adrenergic antagonist phentolamine, suggesting that they might be mediated by β‐adrenergic receptors. Our results indicate for the first time that PPARγ expression can be modulated by the cAMP signalling pathway, and suggest that the anti‐inflammatory effects of NA on brain cells may be partly mediated by increasing PPARγ levels. Conversely, decreased NA due to LC cell death in AD may reduce endogenous PPARγ expression and therefore potentiate neuroinflammatory processes.

Inhibitory and Stimulatory Effects of Lactacystin on Expression of Nitric Oxide Synthase Type 2 in Brain Glial Cells THE ROLE OF IκB-β

Expression of inflammatory nitric oxide synthase (NOS2) is mediated by transcription factor NFκB. By using the specific proteasome inhibitor lactacystin to examine IκB degradation, we observed a paradoxical increase in lipopolysaccharide- and cytokine-dependent NOS2 expression at low concentrations or when lactacystin was added subsequent to cytokines. Lactacystin reduced the initial accumulation of NOS2 mRNA but reduced its subsequent decrease. Lactacystin increased NOS2 promoter activation after 24 h, but not after 4 h, and similarly prevented initial NFκB activation and at later times caused NFκB reactivation. Lactacystin reduced initial degradation of IκB-α and IκB-β, however, at later times selectively increased IκB-β, which was predominantly non-phosphorylated. Expression of full-length rat IκB-β, but not a carboxyl-terminal truncated form, inhibited NOS2 induction and potentiation by lactacystin. Lactacystin increased IκB-β expression in the absence of NOS2 inducers, as well as expression of heat shock protein 70, and the heat shock response due to hyperthermia increased IκB-β expression. These results suggest that IκB-β contributes to persistent NFκB activation and NOS2 expression in glial cells, that IκB-β is a stress protein inducible by hyperthermia or proteasome inhibitors, and that delayed addition of proteasome inhibitors can have stimulatory rather than inhibitory actions.

Local anesthetics potentiate nitric oxide synthase type 2 expression in rat glial cells.

Expression of the calcium-independent nitric oxide synthase (NOS2) contributes to damage in neurologic disease and trauma. The effects of local anesthetics on NOS2 expression have not been examined. The authors tested the effects of four local anesthetics on the expression of NOS2 in immunostimulated rat C6 glioma cells. Incubation with local anesthetics alone did not induce nitrite accumulation; however, the nitrite production induced by stimulation with bacterial endotoxin lipopolysaccharide (LPS) and interferon-&ggr; (IFN-&ggr;) was increased in a dose-dependent manner by bupivacaine (maximal 3-fold at 360 &mgr;M), tetracaine (maximal 7-fold at 360 &mgr;M), and lidocaine at higher doses (5-fold increase at 3.3 mM). Significant increases in nitrite production were observed in concentrations of bupivacaine or tetracaine as low as 120 &mgr;M, which correspond to 30 &mgr;g/mL (.003% weight/volume). In contrast, ropivacaine had little effect on nitrite production (160% of control values) and only at the highest concentration (3.3 mM, corresponding to 890 &mgr;g/mL or 0.089% w/v) tested. Increased nitrite production was not caused by cytotoxic effects of the drugs used, as assessed by release of intracellular lactate dehydrogenase. Increased nitrite production was accompanied by increased NOS2 catalytic activity, steady state mRNA levels, and promoter activation. These results demonstrate that submillimolar doses of two commonly used local anesthetics can increase glial NOS2 expression.