Objective: The pro-inflammatory cytokine interleukin (IL)-1β contributes to the pathogenesis of diverse inflammatory diseases. An example of high clinical relevance is life-threatening systemic inflammation caused by accidental trauma or major surgery that is inter alia induced by extracellular ATP originating from damaged cells. Extracellular ATP activates the P2X7 receptor (P2X7R) and thereby triggers the maturation and release of IL-1β. Therefore, mechanisms controlling ATP-mediated IL-1β release are of substantial clinical interest. We identified a cholinergic mechanism that inhibits the damage-mediated release of IL-1β by human monocytes via flux-independent signaling of nicotinic acetylcholine receptors (nAChRs) containing subunits α7, α9 and/or α10 [1]. Stimulation of nAChRs activates endothelial NO synthase and inhibits the P2X7R by cysteine nitrosylation. Here, we investigate if the lipid raft-associated flotillins are involved in this pathway.

Methods: Human monocytic THP-1 cells, THP-1 cell-derived macrophages, murine peripheral blood mononuclear cells (PBMCs), and murine bone marrow-derived macrophages (BMDMs) isolated from wild-type and flotillin-1 (Flot1) and/or -2 (Flot2) gene-deficient mice were primed with lipopolysaccharide. The ATP-induced release of IL-1β was studied in the presence and the absence of classical (e.g. acetylcholine) and unconventional (e.g. phosphocholine) nAChR agonists. To test for the involvement of nAChRs, the conopeptides [V11L;V16D]ArIB (specific for subunit α7) and RgIA4 (specific for subunits α9/α10) were used. Animals received humane care according to NIH “Guide for the Care and Use of Laboratory Animals”.

Results: Classical and unconventional nAChR agonists efficiently inhibited the ATP-mediated release of IL-1β by monocytic cells and exert similar functions in THP-1 cell-derived macrophages and BMDMs (n ≥ 5, P ≤ 0.05, Friedman followed by Wilcoxon signed-rank test). This inhibitory effect was reversed by the specific conopeptides indicating an involvement of nAChR subunits α7, α9 and/or α10 in mononuclear phagocytes. When investigating the ATP-induced IL-1β release by murine PBMCs and BMDMs, classical and unconventional nAChR agonists efficiently inhibited the ATP-induced IL-1β release by cells from wild-type mice (n ≥ 5, P ≤ 0.05, Friedman followed by Wilcoxon signed-rank test). This inhibitory effect was completely reversed in in mice with a double-deficiency in Flot1 and Flot2 and partially reversed in cells from either Flot1 or Flot2 gene-deficient mice (n ≥ 5, P ≤ 0.05, Kruskal Wallis followed by Mann Whitney rank sum test).

Conclusion: We provide first evidence that the lipid raft-related proteins flotillin-1 and -2 are required for the cholinergic control of ATP-mediated IL-1β release by mononuclear phagocytes. Flotillins have been shown to be associated with several signal transduction pathways. Their exact physiological function is, however, at best partially understood. In the light of the high clinical relevance of the control of ATP-mediated IL-1β release, the exact molecular mechanisms of the interaction of nAChRs, the endothelial NO synthase, the P2X7R, and flotillins deserve further investigation.