gms | German Medical Science

Introduction: Interleukin-1β (IL-1β) is a potent cytokine of innate immunity, which plays a central role in host defense against infections. High systemic concentrations of IL-1β, however, are involved in the pathogenesis of life-threatening systemic inflammatory diseases. Hence, release of IL-1β is typically controlled by two consecutive danger signals. The bacterial cell wall component lipopolysaccharide (LPS) can be the first signal inducing the synthesis of pro-IL-1β. Extracellular ATP released from damaged host cells is a prototypical second danger signal. It is sensed by the ATP receptor P2X7 and leads to inflammasome activation, cleavage of pro-IL-1β, and eventually release of mature IL-1β. β-Nicotinamide adenine dinucleotide (β-NAD) is a well known co-enzyme for redox reactions. In addition, extracellular β-NAD functions as a neurotransmitter leading to relaxation of smooth muscle cells and seems to modulate immune reactions. Typical receptors for β-NAD are P2Y1 and P2Y11. In this study, we test the hypothesis that β-NAD regulates ATP-induced release of IL-1β from LPS-primed monocytes.

Material and methods: U937 cells, a human monocytic cell line, were primed with LPS for 5 hours followed by stimulation with BzATP, a specific ligand for receptor P2X7, in the presence or absence of β-NAD. Release of IL-1β into the cell culture supernatant was measured by ELISA 30 minutes later, along with lactate dehydrogenase to estimate cell death. Receptors involved in effects provoked by β-NAD were identified by the use of specific receptor antagonists (P2Y1: MRS2279; P2Y11: NF340) as well as by siRNA technology. In addition, antagonists of nicotinic acetylcholine receptors were used. The efficiency of gene silencing was controlled by Western blotting.

Results: Stimulation of LPS-primed U937 cells with BzATP induced release of IL-1β into the cell culture medium. IL-1β release was dose-dependently suppressed by addition of β-NAD (IC₅₀ » 50 µM). The suppressive effect of β-NAD was sensitive to NF340, whereas MRS2279 was less effective. Downregulation of P2Y11 expression markedly attenuated the suppressive capacity of β-NAD on the release of IL-1β. In addition, antagonists of nicotinic receptor subunits α7, α9 and α10 completely reversed the effect of β-NAD and enabled BzATP-induced release of IL-1β despite its presence.

Conclusion: We describe a novel antiinflammatory function of extracellular β-NAD, which efficiently suppresses ATP-induced release of IL-1β from human monocytic cells. This effect is mediated by the P2Y11 receptor. P2Y11 activation seems to provoke the release of a yet unknown agonist of nicotinic receptors, which in turn efficiently suppresses ATP-dependent release of IL-1β from human monocytes. A cholinergic mechanism controlling release of IL-1β was recently described by our group. Considering the high clinical relevance of IL-1β, our data may lead to new therapeutic strategies controlling severe inflammation.