ß2-adrenergic agonists modulate TNF-α induced astrocytic inflammatory gene expression and brain inflammatory cell populations.

BACKGROUND: The NF-κB signaling pathway orchestrates many of the intricate aspects of neuroinflammation. Astrocytic ß2-adrenergic receptors have emerged as potential regulators in central nervous system inflammation and are potential targets for pharmacological modulation. The aim of this study was to elucidate the crosstalk between astrocytic ß2-adrenergic receptors and the TNF-α induced inflammatory gene program. METHODS: Proinflammatory conditions were generated by the administration of TNF-α. Genes that are susceptible to astrocytic crosstalk between ß2-adrenergic receptors (stimulated by clenbuterol) and TNF-α were identified by qPCR-macroarray-based gene expression analysis in a human 1321 N1 astrocytoma cell line. Transcriptional patterns of the identified genes in vitro were validated by RT-PCR on the 1321 N1 cell line as well as on primary rat astrocytes. In vivo expression patterns were examined by intracerebroventricular administration of clenbuterol and/or TNF-α in rats. To examine the impact on the inflammatory cell content of the brain we performed extensive FACS analysis of rat brain immune cells after intracerebroventricular clenbuterol and/or TNF-α administration. RESULTS: Parallel transcriptional patterns in vivo and in vitro confirmed the relevance of astrocytic ß2-adrenergic receptors as modulators of brain inflammatory responses. Importantly, we observed pronounced effects of ß2-adrenergic receptor agonists and TNF-α on IL-6, CXCL2, CXCL3, VCAM1, and ICAM1 expression, suggesting a role in inflammatory brain cell homeostasis. Extensive FACS-analysis of inflammatory cell content in the brain demonstrated that clenbuterol/TNF-α co-administration skewed the T cell population towards a double negative phenotype and induced a shift in the myeloid brain cell population towards a neutrophilic predominance. CONCLUSIONS: Our results show that astrocytic ß2-adrenergic receptors are potent regulators of astrocytic TNF-α-activated genes in vitro and in vivo, and ultimately modulate the molecular network involved in the homeostasis of inflammatory cells in the central nervous system. Astrocytic ß2-adrenergic receptors and their downstream signaling pathway may serve as potential targets to modulate neuroinflammatory responses.

Cyclic AMP: a selective modulator of NF-κB action.

It has been known for several decades that cyclic AMP (cAMP), a prototypical second messenger, transducing the action of a variety of G-protein-coupled receptor ligands, has potent immunosuppressive and anti-inflammatory actions. These actions have been attributed in part to the ability of cAMP-induced signals to interfere with the function of the proinflammatory transcription factor Nuclear Factor-kappaB (NF-κB). NF-κB plays a crucial role in switching on the gene expression of a plethora of inflammatory and immune mediators, and as such is one of the master regulators of the immune response and a key target for anti-inflammatory drug design. A number of fundamental molecular mechanisms, contributing to the overall inhibitory actions of cAMP on NF-κB function, are well established. Paradoxically, recent reports indicate that cAMP, via its main effector, the protein kinase A (PKA), also promotes NF-κB activity. Indeed, cAMP actions appear to be highly cell type- and context-dependent. Importantly, several novel players in the cAMP/NF-κB connection, which selectively direct cAMP action, have been recently identified. These findings not only open up exciting new research avenues but also reveal novel opportunities for the design of more selective, NF-κB-targeting, anti-inflammatory drugs.

IL-1ß potently stabilizes IL-6 mRNA in human astrocytes.

Uncontrolled expression of IL-6 in the central nervous system is associated with neurodegenerative pathology and glioma development. Astrocytes are the predominant source of IL-6 in the central nervous system, and they are characteristically susceptible to synergistic IL-6 expression. Combined ß-adrenergic and TNF-receptor triggering induces synergistic IL-6 expression in 1321N1 cells via a transcriptional enhancer mechanism. Here, we have investigated the molecular basis of the very potent "super"-synergistic IL-6 expression that is apparent after combined treatment of astrocytes with a ß-adrenergic agonist, isoproterenol, and the inflammatory cytokines TNF-α and IL-1ß. We found that IL-1ß treatment strengthens the IL-6 synergy by inducing a distinct stabilization of IL-6 mRNA. Surprisingly, the mRNA-stabilizing effect seems to be dependent on protein kinase C (PKC), but not on the prototypical mRNA-stabilizing kinase p38. Moreover, although the mRNA-binding protein HuR basally stabilizes IL-6 mRNA, the mRNA-stabilizing effect of IL-1ß is independent of HuR. Our data using pharmacological inhibitors suggest PKC is an important modulator of IL-6 expression in the central nervous system and this might have therapeutic implications.

Interleukin-6, a mental cytokine.

Almost a quarter of a century ago, interleukin-6 (IL-6) was discovered as an inflammatory cytokine involved in B cell differentiation. Today, IL-6 is recognized to be a highly versatile cytokine, with pleiotropic actions not only in immune cells, but also in other cell types, such as cells of the central nervous system (CNS). The first evidence implicating IL-6 in brain-related processes originated from its dysregulated expression in several neurological disorders such as multiple sclerosis, Alzheimer's disease and Parkinson's disease. In addition, IL-6 was shown to be involved in multiple physiological CNS processes such as neuron homeostasis, astrogliogenesis and neuronal differentiation. The molecular mechanisms underlying IL-6 functions in the brain have only recently started to emerge. In this review, an overview of the latest discoveries concerning the actions of IL-6 in the nervous system is provided. The central position of IL-6 in the neuroinflammatory reaction pattern, and more specifically, the role of IL-6 in specific neurodegenerative processes, which accompany Alzheimer's disease, multiple sclerosis and excitotoxicity, are discussed. It is evident that IL-6 has a dichotomic action in the CNS, displaying neurotrophic properties on the one hand, and detrimental actions on the other. This is in agreement with its central role in neuroinflammation, which evolved as a beneficial process, aimed at maintaining tissue homeostasis, but which can become malignant when exaggerated. In this perspective, it is not surprising that 'well-meant' actions of IL-6 are often causing harm instead of leading to recovery.

Hunting for serine 276-phosphorylated p65.

The transcription factor nuclear factor kappaB (NF-kappaB) is one of the central mediators of inflammatory gene expression. Several posttranslational modifications of NF-kappaB, regulating its transactivation ability, have been described. Especially phosphorylation of the NF-kappaB subunit p65 has been investigated in depth and several commercial phosphospecific antibodies, targeting selected p65 residues, are available. One of the p65 residues, that is subject to phosphorylation by protein kinase A (PKA) as well as by mitogen-stimulated kinase-1 (MSK-1), is the serine at position 276. Here, we have performed a detailed analysis of the performance of the most commonly used commercial anti-P-p65 Ser276 antibodies. Our findings indicate that at least three widely used anti-P-p65 Ser276 antibodies do not detect p65 in vivo via Western Blot, but instead crossreact with PKA-regulated proteins. As PKA is one of the main kinases responsible for phosphorylation of p65 at Ser276, this observation warrants cautious interpretation of data generated using the tested antibodies.

Astrocytic beta(2)-adrenergic receptors: from physiology to pathology.

Evidence accumulates for a key role of the beta(2)-adrenergic receptors in the many homeostatic and neuroprotective functions of astrocytes, including glycogen metabolism, regulation of immune responses, release of neurotrophic factors, and the astrogliosis that occurs in response to neuronal injury. A dysregulation of the astrocytic beta(2)-adrenergic-pathway is suspected to contribute to the physiopathology of a number of prevalent and devastating neurological conditions such as multiple sclerosis, Alzheimer's disease, human immunodeficiency virus encephalitis, stroke and hepatic encephalopathy. In this review we focus on the physiological functions of astrocytic beta(2)-adrenergic receptors, and their possible impact in disease states.

Cooperation of NFkappaB and CREB to induce synergistic IL-6 expression in astrocytes.

Astrocytes are critical players in the innate immune response of the central nervous system. Upon encountering proinflammatory stimuli, astrocytes produce a plethora of inflammatory mediators. Here, we have investigated how beta(2)-adrenergic receptor activation modulates proinflammatory gene expression in astrocytes. We have observed that treatment of human 1321N1 astrocytes with the beta-adrenergic agonist isoproterenol synergistically enhanced TNF-alpha-induced expression of the cytokine IL-6. The effect of isoproterenol was cAMP-dependent and mediated by the beta(2)-adrenergic subtype. Using pharmacological inhibitors and siRNA we showed that protein kinase A (PKA) is an indispensable mediator of the synergy. Simultaneous induction with isoproterenol and TNF-alpha was moreover associated with combined recruitment of CREB and p65 to the native IL-6 promoter. The role of CREB and NFkappaB in promoting the synergy was corroborated using IL-6 promoter point mutants, as well as via siRNA-mediated silencing of CREB and NFkappaB. Interestingly, whereas CREB and NFkappaB usually compete for the limiting cofactor CREB binding protein (CBP), we detected enhanced recruitment of CBP at the IL-6 promoter in our system. The transcriptional synergy seems to be a gene specific process, occurring at the IL-6 and COX-2 gene, but not at other typical NFkappaB-dependent genes such as IL-8, ICAM-1 or VCAM-1. As astrocytic IL-6 overexpression has been associated with neuroinflammatory and neurodegenerative processes, our findings might have important physiological consequences.

Resistance of the dopamine D4 receptor to agonist-induced internalization and degradation.

Dopamine receptors are G-protein-coupled receptors involved in the control of motivation, learning, and fine-tuning of motor movement, as well as modulation of neuroendocrine signalling. Stimulation of G-protein-coupled receptors normally results in attenuation of signalling through desensitization, followed by internalization and down-regulation of the receptor. These processes allow the cell to regain homeostasis after exposure to extracellular stimuli and offer protection against excessive signalling. Here, we have investigated the agonist-mediated attenuation properties of the dopamine D4 receptor. We found that several hallmarks of signal attenuation such as receptor phosphorylation, internalization and degradation showed a blunted response to agonist treatment. Moreover, we did not observe recruitment of beta-arrestins upon D4 receptor stimulation. We also provide evidence for the constitutive phosphorylation of two serine residues in the third intracellular loop of the D4 receptor. These data demonstrate that, when expressed in CHO, HeLa and HEK293 cells, the human D4 receptor shows resistance to agonist-mediated internalization and down-regulation. Data from neuronal cell lines, which have been reported to show low endogenous D4 receptor expression, such as the hippocampal cell line HT22 and primary rat hippocampal cells, further support these observations.