Previous publications demonstrated that activation of β2-adrenergic receptors enhances TNF-α-induced expression of IL-6 in both rat astrocytes and the human 1321 N1 astrocytoma cell line
[15, 23]. This was confirmed in our in vitro qPCR data. Confirmation of these effects in vivo has been lacking. We show, for the first time, by ICV administration of both TNF-α and clenbuterol in rats, that this also occurs in vivo. IL-6 plays an ambiguous role in the CNS, with neurotrophic and neuroprotective effects on the one hand, and destructive effects inducing demyelination and astrogliosis on the other
. Our results apparently contradict a previous study suggesting a suppressive effect of β2-adrenergic receptor activation on IL-6 expression in vivo, as witnessed by decreased IL-6 expression in astrocytes after locus coeruleus lesioning
, as well as a more recent study showing that clenbuterol suppresses IL-6 expression in rat cortex after systemic lipopolysaccharide (LPS) administration
. In the first study there was no pro-inflammatory environment. In the latter study, in which LPS as well as clenbuterol were administered systemically, results might have been confounded by indirect systemic effects which were excluded by our ICV approach. Indeed, systemic administration of LPS increased plasma IL-6, an effect counteracted by intraperitoneal clenbuterol administration
. In addition, although both LPS and TNF-α use NF-κB as an essential signaling mediator, they also induce non-redundant signaling cascades that might explain differences in the outcome of crosstalk with other signaling cascades.
In accordance with the previously described anti-inflammatory action of β-adrenergic receptor agonists, we found that TNF-α-induced expression of ICAM1 and VCAM1 adhesion molecules was antagonized by clenbuterol co-treatment in vitro. However, this inhibitory effect could not be demonstrated in vivo. One possibility is that local inhibitory responses occur in vivo, but that these are masked by the expression of ICAM1 and VCAM1 by cells that do not respond to the clenbuterol treatment in the same manner as astrocytes. A recent study documented a suppressive action of noradrenaline reuptake inhibitors on CAM expression in vivo, which was due to increased noradrenaline availability at glial cells
, suggesting a potential role in regulating inflammatory cell migration across the blood brain barrier. Our data point to the astrocytic β2-adrenergic receptors as possible effectors of noradrenaline action in regulating inflammatory cell migration across the blood brain barrier.
One of the most remarkable findings of this study was the susceptibility of different chemokines to TNF-α/β2-adrenergic receptor interaction in vitro and in vivo. These chemokines have a specific tropism for attracting immune cells. CXCL2 mainly attracts polymorphonuclear leukocytes, CXCL3 controls migration and adhesion of monocytes, and CCL5 is chemotactic for T cells, eosinophils, and basophils
Although it has been previously reported that β-adrenergic receptor activation inhibits NF-κB activity by enhancing the expression of the NF-κB inhibitor IκB in astrocytes
, it is difficult to reconcile such a global NF-κB inhibitory mechanism with our data, showing gene-selective effects of β-agonists. In line with this, we reported that, in 1321 N1 astrocytes, the expression of selected NF-κB target genes was inhibited without apparent changes in IκB levels, indicating additional regulatory mechanisms must exist
. Multiple studies have indicated that activation of the 3′-5′-cyclic adenosine monophosphate (cAMP) – protein kinase A (PKA) – cAMP response element binding protein (CREB) pathway, which is the canonical signaling cascade induced by β-agonists, leads to the competition of active CREB with NF-κB for the limiting co-activator CREB-binding protein (CBP). As CREB has a higher affinity for CBP than NF-κB, the result of this is that the expression of NF-κB target genes, which use CBP as a cofactor, is inhibited upon activation of CREB
. Selected NF-κB target genes, such as IL-6
 and CXC chemokines
, have binding sites for both CREB and NF-κB in their promoters and these genes appear to be targets for potentiation, rather than inhibition, by β-agonists. The exact molecular details of the selective regulation of NF-κB target genes by β-agonists will, however, require further study.
It has been suggested that TNF-α plays an important role in attracting leukocytes towards the brain in diseases as diverse as stroke, HIV-encephalitis, and MS
[32–36]. Astrocytic NF-κB has been shown to play a major role in chemokine-dependent attraction of leukocytes as a result of traumatic brain injury
. In EAE experiments, it has been demonstrated that astrocytic NF-κB modulates chemokine, cytokine and adhesion-molecule expression, CNS inflammatory cell migration, and ultimately clinical outcome
. Since all of the studied NF-κB dependent molecules in our experiments have pleiotropic effects on the myriad of resident brain and immune cells, it is impossible to predict the exact outcome of interventions on neuroinflammatory cell populations. It is, however, remarkable that TNF-α/clenbuterol co-administration shifts the myeloid brain cell population towards a neutrophilic predominance. This correlates well with the in vitro and in vivo potentiation of TNF-α/clenbuterol co-treatment on astrocytic CXCL2 expression that we observed. CXCL2 is a powerful chemo-attractant drawing neutrophils towards the CNS
[38–40]. Astrocytes have been identified as a source of CXCL2 involved in CNS neutrophil migration during early inflammatory responses in mouse spinal cord injury
Another prominent finding in our FACS data is that TNF-α/clenbuterol co-treatment results in a shift towards CD4-CD8- double negative (DN) T cells, expanding from 11% (sham) to 43% (TNF-α/clenbuterol) of the T cell population. These enigmatic cells have been identified as a marginal population in mice and humans, comprising about 1% to 3% of the total T cell pool
[42, 43]. DNT cell prevalence seems to be organ- and inflammation-dependent (for review see
). This subset is thought to act as a regulatory T cell population implicated in counteracting allograft rejection, graft-versus-host disease, and autoimmune processes
[45, 46]. The origins and activation mechanisms of this peculiar subset remain unclear, although it has been shown that extrathymic conversion from CD4+ T cells
 can give rise to potent immunoregulatory DNT cells and CD8+ T cells that have the ability to convert to a DN phenotype
The importance of the shift in neutrophilic predominance over macrophages is more difficult to interpret. Although crucial for clearance of infectious agents, their role in neuroinflammatory conditions remain unclear. In EAE, neutrophil depletion seems to have a protective effect
, although neutrophils are not detected in MS lesions
. In neuromyelitis optica, an antibody-mediated inflammatory condition mainly affecting spinal cord and optic nerves, neutrophils are abundant in lesion pathology
. Recent data from stroke research
 suggest that neutrophils may also have a neuroprotective phenotype (the so called “N2”-phenotype) and that this phenotype may be stimulated by the NF-κB inhibitory pathway PPAR-γ
. The potential of NF-κB inhibition in inducing neuroprotective neutrophils deserves further attention.
To the best of our knowledge, we are the first to describe the presence of the DNT subset in rat brain and its upregulation by TNF-α and β2-adrenergic receptor co-treatment. We previously reported that β2-adrenergic receptors are selectively downregulated in astrocytes in MS
. This downregulation might play a role in the neurodegenerative aspect of the disease
, but it remains unclear how it can explain the inflammatory aspect of MS. A decrease in immunoregulatory DNT cells may be a component linking downregulation of astrocytic β2-adrenergic receptors with neuroinflammation in MS. It should be stressed that these findings are preliminary and that the mechanisms behind these shifts in T cell subsets and the potential role of β2-adrenergic receptors in regulating CNS autoimmunity deserve further investigation.