Auto/paracrine control of inflammatory cytokines by acetylcholine in macrophage-like U937 cells through nicotinic receptors

Abstract

Although acetylcholine (ACh) is well known for its neurotransmitter function, recent studies have indicated that it also functions as an immune cytokine that prevents macrophage activation through a ‘cholinergic (nicotinic) anti-inflammatory pathway’. In this study, we used the macrophage-like U937 cells to elucidate the mechanisms of the physiologic control of cytokine production by auto/paracrine ACh through the nicotinic class of ACh receptors (nAChRs) expressed in these cells. Stimulation of cells with lipopolysaccharide up-regulated expression of α1, α4, α5, α7, α10, β1 and β3 subunits, down-regulated α6 and β2 subunits, and did not alter the relative quantity of α9 and β4 mRNAs. Distinct nAChR subtypes showed differential regulation of the production of pro- and anti-inflammatory cytokines. While inhibition of the expression of the TNF-α gene was mediated predominantly by the α-bungarotoxin sensitive nAChRs, that of the IL-6 and IL-18 genes—by the mecamylamine-sensitive nAChRs. Both the Mec- and αBtx-sensitive nAChRs regulated expression of the IL-1β gene equally efficiently. Upregulation of IL-10 production by auto/paracrine ACh was mediated predominantly through α7 nAChR. These findings offer a new insight on how nicotinic agonists control inflammation, thus laying a groundwork for the development of novel immunomodulatory therapies based on the nAChR subtype selectivity of nicotinic agonists.

Introduction

Although acetylcholine (ACh) is well known for its neurotransmitter function, recent studies have indicated that it also functions as an immune cytokine that prevents macrophage activation through a ‘cholinergic (nicotinic) anti-inflammatory pathway’ [1], [2]. The nicotinic ACh receptor (nAChR) agonists have been shown to prevent or treat experimentally induced endotoxemic shock [3], [4], [5], sepsis [5], [6], [7], hemorrhagic shock [8], [9], ischemia-reperfusion [10], subcutaneous inflammation [11], postoperative ileus [12], pancreatitis [13], allergic lung inflammation [14], [15], and acute lung injury [16]. The agonist of nAChRs nicotine has been used in clinical trials, but its clinical potential is limited by its collateral toxicity [17]. Appreciations of an important role of α7 nAChR in regulation of the immune inflammation urged a search for selective nicotinic agonists that avoid the undesired side effects of nicotine [2]. Further elucidation of the nAChR-mediated regulation of inflammation should help develop novel treatments allowing to regulate specific types of immune reactions by selectively activating or blocking particular nAChR subtypes expressed in monocytes/macrophages.

Various immune cells possess diverse repertoires of nAChRs and, therefore, respond differently to the nicotinic agonists that exhibit varying affinities to distinct nAChR subtypes. The pharmacologic subtype of the ACh-gated ion channel is determined by a specific combination of the nAChR subunits forming the channel. The “muscle”-type nAChRs can be comprised by α1, β1, γ, δ, and ε subunits, and the “neuronal”-type nAChRs—by α2–α10 and β2–β4 subunits [18], [19], [20], [21]. The α7, and α9 subunits can form homomeric nAChR channels sensitive to α-bungarotoxin (αBtx). The heteromeric channels can be composed of α2, α3, α4, α5, α6, β2, β3 and β4 subunits, e.g., α3(β2/β4) ± α5, and α9 can also form a heteromeric channel with α10 [21]. The signal transduction pathways downstream of different nAChRs may be activated by both ionic events, such as Ca²⁺ influx, and changes of the stoichiometry of a multiprotein complex formed by the nAChR subunit(s) [22], [23]. Therefore, a net biologic effect of ACh in a particular type of immune cell depends on the subunit composition of the major nAChR subtypes expressed by the cell at a given stage of its development and activation.

The presence of nAChRs in human monocyte/macrophages was suggested by the inhibitory effect of αBtx on monocyte activation [24] and nicotine binding to the human monocytic THP-1 cell line [25]. By now, it has been documented that human, murine and monkey macrophages express classic nAChR subunits [4], [26], [27]. Expression of α1, α7, and α10 mRNAs has been detected in human macrophages [4], whereas both bone marrow-derived dendritic cells and macrophages from C57BL/6J mice possess mRNAs encoding the nAChR subunits α2, α5, α6, α7, α10 and β2 [28]. Macrophages also express the muscarinic class of ACh receptors [29], [30] that can modify the cell response to auto/paracrine ACh.

The human monoblastoid tumor cell line U937 [31] that can be differentiated into macrophage-like cells by treatment with phorbol-12-myristate 13-acetate (PMA) exhibits ACh synthesizing activity of choline acetyltransferase and contains approximately 0.02 pmol/10⁶ of ACh [32]. Although, to the best of our knowledge, the subunit composition of nAChRs expressed in U937 cells have not been established, it has been reported that these cells respond to nicotine [33], [34]. Therefore, U937 cells provide a useful model for studying basic mechanisms of macrophage regulation by auto/paracrine ACh through nAChRs.

In this study, we characterized the profile of nAChR subunits expressed in the macrophage-like differentiated U937 cells and demonstrated how the receptor repertoire changes upon cell activation with lipopolysaccharide (LPS). We also established relative contributions of α7- and non-α7 nAChR subtypes expressed in these cells to regulation of the pro- and anti-inflammatory cytokine production. The obtained results indicated that the macrophage nAChR subtypes are differentially coupled to regulation of production of distinct cytokines by auto/paracrine ACh. These findings offer a new insight on how nicotinic agonists control inflammation, thus laying a groundwork for the development of novel immunomodulatory therapies based on the nAChR subtype selectivity of nicotinic agonists.