Investigating the role of α7 nicotinic receptors in inflammation

Alpha7 nicotinic acetylcholine receptors (α7 nAChRs) are ligand-gated ion-channels located in brain, retina, autonomic ganglia and some immune cells such as macrophages. α7 nAChRs have emerged as a novel target to inhibit peripheral inflammation by blocking the transcription factor, nuclear factor kappa-light chain-enhancer of B cells (NFΚB). α7 nAChR agonists including nicotine and GTS-21 have shown great promise in reducing levels of the pro-inflammatory cytokines such as interleukin-6 (IL6) and tumor-necrosis factor (TNF) in murine endotoxemia and severe sepsis models in vivo. However, mechanistic aspects of GTS-21effects on inflammatory pathways are largely unexplored. Here we used GH4C1 cells overexpressing α7 nAChR to evaluate the effects of nicotine and GTS-21 on TNF-induced NFΚB signaling. We also compared cell-type dependent responses of both α7 nAChR agonists in GH4C1 cells (non-immune cells), RAW264.7 cells (macrophage-like immune cells) and in ex vivo cultures of primary mouse macrophages. Nicotine and GTS-21 do not block TNF-stimulated NFΚB signaling in GH4C1 cells overexpressing α7 nAChRs, suggesting that they require additional unidentified factors, other than α7 nAChRs, for this anti-inflammatory effect. GTS-21 dose-dependently suppressed lipopolysaccharide (LPS)-induced TNF secretion in RAW264.7 cells lacking α7 nAChRs and primary mouse macrophages expressing them. In contrast, GTS-21 blocked IL6 secretion in primary mouse macrophages but had no effect in RAW264.7 cells. Similarly, nicotine inhibited LPS-activated IL6 and TNF secretion in primary mouse macrophages; however it had no effect on endotoxin-activated RAW264.7 cells. Moreover, α7 nAChR antagonism, using either methyllycaconitine (MLA) or α-bungarotoxin (αBGT), partially reversed nicotine or GTS-21 blockade of IL6 and TNF secretion in primary mouse macrophages. Since anti-inflammatory effects of nicotine were smaller than those of GTS-21 in mouse macrophages and because α7 nAChR involvement in nicotine's effects have been well characterized in macrophages, we used α7 nAChR-knockout (Chrna7-/-) mouse models to measure the importance of α7 nAChR in GTS-21 mediated anti-inflammatory effects. GTS-21 significantly inhibited LPS-induced IL6 and TNF production in Chrna7-/- mouse macrophages. These data indicate that even though a component of the GTS-21 anti-inflammatory effects requires an α7 nAChR presence, GTS-21 also has anti-inflammatory effects independent of α7 nAChR receptors, and these effects depend on the cell type.; Because α7 nAChRs are implicated in several disorders such as Alzheimer's, Parkinson's, schizophrenia, and inflammation and are involved in memory and cognition, validated methods to detect and localize these receptors are highly desirable. However, questions have been raised about the utility of commercially available α7 nAChR antibodies to detect rat α7 nAChRs. We therefore developed a gel-shift assay for western blots using GH4C1 cells overexpressing rat α7 nAChR and rat α7 nAChR-green fluorescent protein (GFP) construct…