Inhibition of microglial inflammatory responses by norepinephrine: effects on nitric oxide and interleukin-1β production

Background: 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-1 beta plays in that response. Methods: 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-1 beta 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 NF kappa B:I kappa B pathway were examined by using selective a NF kappa B inhibitor and measuring I kappa B alpha protein levels by western blots. A role for IL-1 beta in NOS2 induction was tested by examining effects of caspase-1 inhibitors and using caspase-1 deficient cells. Results: LPS caused a time-dependent increase in NOS2 mRNA levels and NO production; which was blocked by a selective NF kappa B inhibitor. NE dose-dependently reduced NOS2 expression and NO generation, via activation of beta 2-adrenergic receptors (beta 2-ARs), and reduced loss of inhibitory IkB alpha 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-1 beta was suggested since NE potently blocked microglial IL-1 beta production. However, incubation with a caspase-1 inhibitor, which reduced IL-1 beta 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. Conclusions: NE reduces microglial NOS2 expression and IL-1 beta production, however IL-1 beta 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.