Recently, we discovered a cholinergic mechanism that inhibits the adenosine triphosphate (ATP)-dependent release of interleukin-1β (IL-1β) by human monocytes via nicotinic acetylcholine receptors (nAChRs) composed of α7, α9 and/or α10 subunits. Furthermore, we identified phosphocholine (PC) and dipalmitoylphosphatidylcholine (DPPC) as novel nicotinic agonists that elicit metabotropic activity at monocytic nAChR. Interestingly, PC does not provoke ion channel responses at conventional nAChRs composed of subunits α9 and α10. The purpose of this study is to determine the composition of nAChRs necessary for nicotinic signaling in monocytic cells and to test the hypothesis that common metabolites of phosphatidylcholines, lysophosphatidylcholine (LPC) and glycerophosphocholine (G-PC), function as nAChR agonists. In peripheral blood mononuclear cells from nAChR gene-deficient mice, we demonstrated that inhibition of ATP-dependent release of IL-1β by acetylcholine (ACh), nicotine and PC depends on subunits α7, α9 and α10. Using a panel of nAChR antagonists and siRNA technology, we confirmed the involvement of these subunits in the control of IL-1β release in the human monocytic cell line U937. Furthermore, we showed that LPC (C16:0) and G-PC efficiently inhibit ATP-dependent release of IL-1β. Of note, the inhibitory effects mediated by LPC and G-PC depend on nAChR subunits α9 and α10, but only to a small degree on α7. In Xenopus laevis oocytes heterologously expressing different combinations of human α7, α9 or α10 subunits, ACh induced canonical ion channel activity, whereas LPC, G-PC and PC did not. In conclusion, we demonstrate that canonical nicotinic agonists and PC elicit metabotropic nAChR activity in monocytes via interaction of nAChR subunits α7, α9 and α10. For the metabotropic signaling of LPC and G-PC, nAChR subunits α9 and α10 are needed, whereas α7 is virtually dispensable. Furthermore, molecules bearing a PC group in general seem to regulate immune functions without perturbing canonical ion channel functions of nAChR.

While interleukin (IL)-1β is a potent pro-inflammatory cytokine involved in host defense, high levels can cause life-threatening sterile inflammation including systemic inflammatory response syndrome. Hence, the control of IL-1β secretion is of outstanding biomedical importance. In response to a first inflammatory stimulus such as lipopolysaccharide, pro-IL-1β is synthesized as a cytoplasmic inactive pro-form. Extracellular ATP originating from injured cells is a prototypical second signal for inflammasome-dependent maturation and release of IL-1β. The human anti-protease alpha-1 antitrypsin (AAT) and IL-1β regulate each other via mechanisms that are only partially understood. Here, we demonstrate that physiological concentrations of AAT efficiently inhibit ATP-induced release of IL-1β from primary human blood mononuclear cells, monocytic U937 cells, and rat lung tissue, whereas ATP-independent IL-1β release is not impaired. Both, native and oxidized AAT are active, suggesting that the inhibition of IL-1β release is independent of the anti-elastase activity of AAT. Signaling of AAT in monocytic cells involves the lipid scavenger receptor CD36, calcium-independent phospholipase A2β, and the release of a small soluble mediator. This mediator leads to the activation of nicotinic acetylcholine receptors, which efficiently inhibit ATP-induced P2X7 receptor activation and inflammasome assembly. We suggest that AAT controls ATP-induced IL-1β release from human mononuclear blood cells by a novel triple-membrane-passing signaling pathway. This pathway may have clinical implications for the prevention of sterile pulmonary and systemic inflammation.

-In pulmonary epithelia, ␤-adrenergic agonists regulate the membrane abundance of the epithelial sodium channel (ENaC) and, thereby, control the rate of transepithelial electrolyte absorption. This is a crucial regulatory mechanism for lung liquid clearance at birth and thereafter. This study investigated the influence of the gaseous signaling molecule hydrogen sulfide (H 2S) on ␤-adrenergic agonistregulated pulmonary sodium and liquid absorption. Application of the H2S-liberating molecule Na2S (50 M) to the alveolar compartment of rat lungs in situ decreased baseline liquid absorption and abrogated the stimulation of liquid absorption by the ␤-adrenergic agonist terbutaline. There was no additional effect of Na2S over that of the ENaC inhibitor amiloride. In electrophysiological Ussing chamber experiments with native lung epithelia (Xenopus laevis), Na 2S inhibited the stimulation of amiloride-sensitive current by terbutaline. ␤-adrenergic agonists generally increase ENaC abundance by cAMP formation and activation of PKA. Activation of this pathway by forskolin and 3-isobutyl-1-methylxanthine increased amiloride-sensitive currents in H441 pulmonary epithelial cells. This effect was inhibited by Na 2S in a dose-dependent manner (5-50 M). Na2S had no effect on cellular ATP concentration, cAMP formation, and activation of PKA. By contrast, Na 2S prevented the cAMP-induced increase in ENaC activity in the apical membrane of H441 cells. H441 cells expressed the H 2S-generating enzymes cystathionine-␤-synthase, cystathionine-␥-lyase, and 3-mercaptopyruvate sulfurtransferase, and they produced H 2S amounts within the employed concentration range. These data demonstrate that H 2S prevents the stimulation of ENaC by cAMP/PKA and, thereby, inhibits the proabsorptive effect of ␤-adrenergic agonists on lung liquid clearance.

Interleukin-1β (IL-1β) is a potent, pro-inflammatory cytokine of the innate immune system that plays an essential role in host defense against infection. However, elevated circulating levels of IL-1β can cause life-threatening systemic inflammation. Hence, mechanisms controlling IL-1β maturation and release are of outstanding clinical interest. Secretory leukocyte protease inhibitor (SLPI), in addition to its well-described anti-protease function, controls the expression of several pro-inflammatory cytokines on the transcriptional level. In the present study, we tested the potential involvement of SLPI in the control of ATP-induced, inflammasome-dependent IL-1β maturation and release. We demonstrated that SLPI dose-dependently inhibits the ATP-mediated inflammasome activation and IL-1β release in human monocytic cells, without affecting the induction of pro-IL-1β mRNA by LPS. In contrast, the ATP-independent IL-1β release induced by the pore forming bacterial toxin nigericin is not impaired, and SLPI does not directly modulate the ion channel function of the human P2X 7 receptor heterologously expressed in Xenopus laevis oocytes. In human monocytic U937 cells, however, SLPI efficiently inhibits ATP-induced ion-currents. Using specific inhibitors and siRNA, we demonstrate that SLPI activates the calcium-independent phospholipase A2β (iPLA2β) and leads to the release of a low molecular mass factor that mediates the inhibition of IL-1β release. Signaling involves nicotinic acetylcholine receptor subunits α7, α9, α10, and Src kinase activation and results in an inhibition of ATP-induced caspase-1 activation. In conclusion, we propose a novel anti-inflammatory mechanism induced by SLPI, which inhibits the ATP-dependent maturation and secretion of IL-1β. This novel signaling pathway might lead to development of therapies that are urgently needed for the prevention and treatment of systemic inflammation.