β₂-Adrenergic receptor regulates Toll-like receptor-4-induced nuclear factor-κB activation through β-arrestin 2

Toll-like receptors (TLRs) play an important role in innate immunity while, β₂-adrenergic receptors (β₂AR) provide the key linkages for the sympathetic nervous system to regulate the immune system. However, their role in macrophages remains uncertain. Here, we demonstrate the cross-talk between β₂AR and TLR signalling pathways. Expression of β₂AR was down-regulated by TLR4 ligand lipopolysaccharide (LPS) stimulation. To investigate the physiological consequence of this down-regulation RAW264 cells, a macrophage cell line, were transfected with a β₂AR expression vector (RAWar). Both LPS-stimulated inducible nitric oxide synthase (NOS II) expression and NO production were markedly suppressed in the RAWar cells. The activation of nuclear factor-κB (NF-κB) and degradation of the inhibitor of NF-κB (IκBα) in response to LPS were markedly decreased in these cells. The level of β-arrestin 2, which regulates β₂AR signalling, was also reduced in RAW264 cells after stimulation with LPS, but not in RAWar cells. Overexpression of β-arrestin 2 (RAWarr2) also inhibited NO production and NOS II expression. Furthermore, we demonstrated that β-arrestin 2 interacted with cytosolic IκBα and that the level of IκBα coimmunoprecipitated by anti-β-arrestin 2 antibodies was decreased in the RAW264 cells but not in RAWar or RAWarr2 cells. These findings suggest that LPS-stimulated signals suppress β₂AR expression, leading to down-regulation of β-arrestin 2 expression, which stabilizes cytosolic IκBα and inhibits the NF-κB activation essential for NOS II expression, probably to ensure rapid and sufficient production of NO in response to microbial attack.