Activation of endothelial NO synthase and P2X7 receptor modification mediates the cholinergic control of ATP-induced interleukin-1ß release by mononuclear phagocytes.

Objective: The pro-inflammatory cytokine interleukin-1ß (IL-1ß) plays a central role in host defense against infections. High systemic IL-1ß levels, however, promote the pathogenesis of inflammatory disorders. Therefore, mechanisms controlling IL-1ß release are of substantial clinical interest. Recently, we identified a cholinergic mechanism inhibiting the ATP-mediated IL-1ß release by human monocytes via nicotinic acetylcholine receptor (nAChR) subunits α7, α9 and/or α10. We also discovered novel nAChR agonists that trigger this inhibitory function in monocytic cells without eliciting ionotropic functions at conventional nAChRs. Here, we investigate the ion flux-independent signaling pathway that links nAChR activation to the inhibition of the ATP-sensitive P2X7 receptor (P2X7R). Methods: Different human and murine mononuclear phagocytes were primed with lipopolysaccharide and stimulated with the P2X7R agonist BzATP in the presence or absence of nAChR agonists, endothelial NO synthase (eNOS) inhibitors, and NO donors. IL-1ß was measured in cell culture supernatants. Patch-clamp and intracellular Ca2+ imaging experiments were performed on HEK cells overexpressing human P2X7R or P2X7R with point mutations at cysteine residues in the cytoplasmic C-terminal domain. Results: The inhibitory effect of nAChR agonists on the BzATP-induced IL-1ß release was reversed in the presence of eNOS inhibitors (L-NIO, L-NAME) as well as in U937 cells after silencing of eNOS expression. In peripheral blood mononuclear leukocytes from eNOS gene-deficient mice, the inhibitory effect of nAChR agonists was absent, suggesting that nAChRs signal via eNOS to inhibit the BzATP-induced IL-1ß release. Moreover, NO donors (SNAP, S-nitroso-N-acetyl-DL-penicillamine; SIN-1) inhibited the BzATP-induced IL-1ß release by mononuclear phagocytes. The BzATP-induced ionotropic activity of the P2X7R was abolished in the presence of SIN-1 in both, Xenopus laevis oocytes and HEK cells over-expressing the human P2X7R. This inhibitory effect of SIN-1 was absent in HEK cells expressing P2X7R, in which C377 was mutated to alanine, indicating the importance of C377 for the regulation of the P2X7R function by protein modification. Conclusion: We provide first evidence that ion flux-independent, metabotropic signaling of monocytic nAChRs involves eNOS activation and P2X7R modification, resulting in an inhibition of ATP signaling and ATP-mediated IL-1ß release. This signaling pathway might be an interesting target for the treatment of inflammatory disorders.

Amyloid Beta Peptide (Aß1-42) Reverses the Cholinergic Control of Monocytic IL-1ß Release.

Amyloid-ß peptide (Aß1-42), the cleavage product of the evolutionary highly conserved amyloid precursor protein, presumably plays a pathogenic role in Alzheimer's disease. Aß1-42 can induce the secretion of the pro-inflammatory cytokine intereukin-1ß (IL-1ß) in immune cells within and out of the nervous system. Known interaction partners of Aß1-42 are α7 nicotinic acetylcholine receptors (nAChRs). The physiological functions of Aß1-42 are, however, not fully understood. Recently, we identified a cholinergic mechanism that controls monocytic release of IL-1ß by canonical and non-canonical agonists of nAChRs containing subunits α7, α9, and/or α10. Here, we tested the hypothesis that Aß1-42 modulates this inhibitory cholinergic mechanism. Lipopolysaccharide-primed monocytic U937 cells and human mononuclear leukocytes were stimulated with the P2X7 receptor agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate triethylammonium salt (BzATP) in the presence or absence of nAChR agonists and Aß1-42. IL-1ß concentrations were measured in the supernatant. Aß1-42 dose-dependently (IC50 = 2.54 µM) reversed the inhibitory effect of canonical and non-canonical nicotinic agonists on BzATP-mediated IL-1ß-release by monocytic cells, whereas reverse Aß42-1 was ineffective. In conclusion, we discovered a novel pro-inflammatory Aß1-42 function that enables monocytic IL-1ß release in the presence of nAChR agonists. These findings provide evidence for a novel physiological function of Aß1-42 in the context of sterile systemic inflammation.

Phosphocholine-Modified Lipooligosaccharides of Haemophilus influenzae Inhibit ATP-Induced IL-1ß Release by Pulmonary Epithelial Cells.

Phosphocholine-modified bacterial cell wall components are virulence factors enabling immune evasion and permanent colonization of the mammalian host, by mechanisms that are poorly understood. Recently, we demonstrated that free phosphocholine (PC) and PC-modified lipooligosaccharides (PC-LOS) from Haemophilus influenzae, an opportunistic pathogen of the upper and lower airways, function as unconventional nicotinic agonists and efficiently inhibit the ATP-induced release of monocytic IL-1ß. We hypothesize that H. influenzae PC-LOS exert similar effects on pulmonary epithelial cells and on the complex lung tissue. The human lung carcinoma-derived epithelial cell lines A549 and Calu-3 were primed with lipopolysaccharide from Escherichia coli followed by stimulation with ATP in the presence or absence of PC or PC-LOS or LOS devoid of PC. The involvement of nicotinic acetylcholine receptors was tested using specific antagonists. We demonstrate that PC and PC-LOS efficiently inhibit ATP-mediated IL-1ß release by A549 and Calu-3 cells via nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10. Primed precision-cut lung slices behaved similarly. We conclude that H. influenzae hijacked an endogenous anti-inflammatory cholinergic control mechanism of the lung to evade innate immune responses of the host. These findings may pave the way towards a host-centered antibiotic treatment of chronic airway infections with H. influenzae.

ß-Nicotinamide Adenine Dinucleotide (ß-NAD) Inhibits ATP-Dependent IL-1ß Release from Human Monocytic Cells.

While interleukin-1ß (IL-1ß) is a potent pro-inflammatory cytokine essential for host defense, high systemic levels cause life-threatening inflammatory syndromes. ATP, a stimulus of IL-1ß maturation, is released from damaged cells along with ß-nicotinamide adenine dinucleotide (ß-NAD). Here, we tested the hypothesis that ß-NAD controls ATP-signaling and, hence, IL-1ß release. Lipopolysaccharide-primed monocytic U937 cells and primary human mononuclear leukocytes were stimulated with 2'(3')-O-(4-benzoyl-benzoyl)ATP trieethylammonium salt (BzATP), a P2X7 receptor agonist, in the presence or absence of ß-NAD. IL-1ß was measured in cell culture supernatants. The roles of P2Y receptors, nicotinic acetylcholine receptors (nAChRs), and Ca2+-independent phospholipase A2 (iPLA2ß, PLA2G6) were investigated using specific inhibitors and gene-silencing. Exogenous ß-NAD signaled via P2Y receptors and dose-dependently (IC50 = 15 µM) suppressed the BzATP-induced IL-1ß release. Signaling involved iPLA2ß, release of a soluble mediator, and nAChR subunit α9. Patch-clamp experiments revealed that ß-NAD inhibited BzATP-induced ion currents. In conclusion, we describe a novel triple membrane-passing signaling cascade triggered by extracellular ß-NAD that suppresses ATP-induced release of IL-1ß by monocytic cells. This cascade links activation of P2Y receptors to non-canonical metabotropic functions of nAChRs that inhibit P2X7 receptor function. The biomedical relevance of this mechanism might be the control of trauma-associated systemic inflammation.

Chemokines (CCL3, CCL4, and CCL5) Inhibit ATP-Induced Release of IL-1ß by Monocytic Cells.

Chemokines and ATP are among the mediators of inflammatory sites that can enter the circulation via damaged blood vessels. The main function of chemokines is leukocyte mobilization, and ATP typically triggers inflammasome assembly. IL-1ß, a potent inflammasome-dependent cytokine of innate immunity, is essential for pathogen defense. However, excessive IL-1ß may cause life-threatening systemic inflammation. Here, we hypothesize that chemokines control ATP-dependent secretion of monocytic IL-1ß. Lipopolysaccharide-primed human monocytic U937 cells were stimulated with the P2X7 agonist BzATP for 30 min to induce IL-1ß release. CCL3, CCL4, and CCL5 dose dependently inhibited BzATP-stimulated release of IL-1ß, whereas CXCL16 was ineffective. The effect of CCL3 was confirmed for primary mononuclear leukocytes. It was blunted after silencing CCR1 or calcium-independent phospholipase A2 (iPLA2) by siRNA and was sensitive to antagonists of nicotinic acetylcholine receptors containing subunits α7 and α9. U937 cells secreted small factors in response to CCL3 that mediated the inhibition of IL-1ß release. We suggest that CCL chemokines inhibit ATP-induced release of IL-1ß from U937 cells by a triple-membrane-passing mechanism involving CCR, iPLA2, release of small mediators, and nicotinic acetylcholine receptor subunits α7 and α9. We speculate that whenever chemokines and ATP enter the circulation concomitantly, systemic release of IL-1ß is minimized.

Canonical and Novel Non-Canonical Cholinergic Agonists Inhibit ATP-Induced Release of Monocytic Interleukin-1ß via Different Combinations of Nicotinic Acetylcholine Receptor Subunits α7, α9 and α10.

Recently, we discovered a cholinergic mechanism that inhibits the adenosine triphosphate (ATP)-dependent release of interleukin-1ß (IL-1ß) 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-1ß 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-1ß 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-1ß. 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 Xenopuslaevis 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.

Surfactant inhibits ATP-induced release of interleukin-1ß via nicotinic acetylcholine receptors.

Interleukin (IL)-1ß is a potent pro-inflammatory cytokine of innate immunity involved in host defense. High systemic IL-1ß levels, however, cause life-threatening inflammatory diseases, including systemic inflammatory response syndrome. In response to various danger signals, the pro-form of IL-1ß is synthesized and stays in the cytoplasm unless a second signal, such as extracellular ATP, activates the inflammasome, which enables processing and release of mature IL-1ß. As pulmonary surfactant is known for its anti-inflammatory properties, we hypothesize that surfactant inhibits ATP-induced release of IL-1ß. Lipopolysaccharide-primed monocytic U937 cells were stimulated with an ATP analog in the presence of natural or synthetic surfactant composed of recombinant surfactant protein (rSP)-C, palmitoylphosphatidylglycerol, and dipalmitoylphosphatidylcholine (DPPC). Both surfactant preparations dose-dependently inhibited IL-1ß release from U937 cells. DPPC was the active constituent of surfactant, whereas rSP-C and palmitoylphosphatidylglycerol were inactive. DPPC was also effective in primary mononuclear leukocytes isolated from human blood. Experiments with nicotinic antagonists, siRNA technology, and patch-clamp experiments suggested that stimulation of nicotinic acetylcholine receptors (nAChRs) containing subunit α9 results in a complete inhibition of the ion channel function of ATP receptor, P2X7. In conclusion, the surfactant constituent, DPPC, efficiently inhibits ATP-induced inflammasome activation and maturation of IL-1ß in human monocytes by a mechanism involving nAChRs.

Phosphocholine-Modified Macromolecules and Canonical Nicotinic Agonists Inhibit ATP-Induced IL-1ß Release.

IL-1ß is a potent proinflammatory cytokine of the innate immune system that is involved in host defense against infection. However, increased production of IL-1ß plays a pathogenic role in various inflammatory diseases, such as rheumatoid arthritis, gout, sepsis, stroke, and transplant rejection. To prevent detrimental collateral damage, IL-1ß release is tightly controlled and typically requires two consecutive danger signals. LPS from Gram-negative bacteria is a prototypical first signal inducing pro-IL-1ß synthesis, whereas extracellular ATP is a typical second signal sensed by the ATP receptor P2X7 that triggers activation of the NLRP3-containing inflammasome, proteolytic cleavage of pro-IL-1ß by caspase-1, and release of mature IL-1ß. Mechanisms controlling IL-1ß release, even in the presence of both danger signals, are needed to protect from collateral damage and are of therapeutic interest. In this article, we show that acetylcholine, choline, phosphocholine, phosphocholine-modified LPS from Haemophilus influenzae, and phosphocholine-modified protein efficiently inhibit ATP-mediated IL-1ß release in human and rat monocytes via nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10. Of note, we identify receptors for phosphocholine-modified macromolecules that are synthesized by microbes and eukaryotic parasites and are well-known modulators of the immune system. Our data suggest that an endogenous anti-inflammatory cholinergic control mechanism effectively controls ATP-mediated release of IL-1ß and that the same mechanism is used by symbionts and misused by parasites to evade innate immune responses of the host.

Adaptive and innate immune responses in a rat orthotopic lung transplant model of chronic lung allograft dysfunction.

Acute rejection and respiratory infections are major risk factors for chronic lung allograft dysfunction (CLAD) after lung transplantation. To shed light on the enigmatic etiology of CLAD, we test the following hypotheses using a new experimental model: (i) Alloimmune-independent pulmonary inflammation reactivates alloimmunity. (ii) Alloimmunity enhances the susceptibility of the graft toward pathogen-associated molecular patterns. Pulmonary Fischer 344 to Lewis rat allografts were treated with lipopolysaccharide (LPS), which consistently results in lesions typical for CLAD. Grafts, local lymph nodes, and spleens were harvested before (day 28) and after LPS application (days 29, 33, and 40) for real-time RT-PCR and immunohistochemistry. Mixed lymphocyte reactions were performed on day 33. Four weeks after transplantation, lung allografts displayed mononuclear infiltrates compatible with acute rejection and overexpressed most components of the toll-like receptor system. Allografts but not secondary lymphoid organs expressed increased levels of Th1-type transcription factors and cytokines. LPS induced macrophage infiltration as well as mRNA expression of pro-inflammatory cytokines and effector molecules of innate immunity. Unexpectedly, T-cell reactivity was not enhanced by LPS. We conclude that prevention of CLAD might be accomplished by local suppression of Th1 cells in stable grafts and by controlling innate immunity during alloimmune-independent pulmonary inflammation.

Expression of peptide YY by human blood leukocytes.

Peptide YY is produced by L cells in the mucosa of the distal ileum, colon, and rectum and may have systemic and paracrine functions. We hypothesized that peptide YY is expressed by peripheral blood mononuclear cells. The purpose of the present study was to evaluate the expression of peptide YY mRNA and peptide by peripheral blood mononuclear cells and differentiated THP-1 cells after lipopolysaccharide treatment as an in vitro model of inflammation. Blood was drawn by venipuncture from 18- to 63-year-old healthy male blood donors (n=63); peptide YY mRNA expression levels were detected in peripheral blood mononuclear cells from all healthy male subjects. In 3 subjects, peripheral blood mononuclear cells were cultured for 3 and 24h and peptide YY was detected in the cell culture supernatant. In human monocytic THP-1 cells treated with phorbol-12-myristate-13-acetate to induce differentiation to macrophages, treatment with lipopolysaccharide caused down-regulation of peptide YY mRNA levels. In summary, freshly isolated peripheral blood mononuclear cells from healthy humans expressed peptide YY. In vitro data suggested that peptide YY expression is down-regulated by differentiation of monocytes to macrophages and proinflammatory stimuli.

Pivotal Advance: Up-regulation of acetylcholine synthesis and paracrine cholinergic signaling in intravascular transplant leukocytes during rejection of rat renal allografts.

During acute rejection, large numbers of leukocytes accumulate in the blood vessels of experimental renal allografts. About 70% of them are activated, cytotoxic monocytes that appear to be involved in allograft destruction. ACh exerts anti-inflammatory effects upon monocytes/macrophages and has been proposed to be a key player in neuroimmunological interactions. Its short half-life, however, makes it unlikely that neuronal ACh affects blood leukocytes. Renal transplantation was performed in the allogeneic DA to LEW and in the isogeneic LEW to LEW rat strain combination. Intravascular leukocytes were harvested after 4 days, and the expression of CHT1, cChAT, pChAT, and nAChR subunits was investigated by RT-PCR, immunoblotting, and immunohistochemistry. Monocytes were identified by double-labeling with ED1-antibody, directed to a CD68-like antigen. ACh content was measured by HPLC. [Ca(2+)](i) was monitored by Fura-2. Intravascular graft leukocytes express CHT1 and cChAT mRNA and protein and pChAT protein. Their expression is strongly up-regulated in vivo during acute allograft rejection. Immunohistochemistry revealed CHT1, cChAT, and pChAT protein in ED1-positive monocytes. The ACh content of allograft intravascular leukocytes was sixfold higher than that of isografts. Intravascular leukocytes express nAChR subunits, and an ATP-induced increase in [Ca(2+)](i) was augmented in vitro by a nAChR inhibitor in allograft but not isograft leukocytes. Intravascular graft leukocytes, among them monocytes, up-regulate non-neuronal ACh synthesis and develop auto-/paracrine cholinergic attenuation of ATP signaling during acute allograft rejection.

Administration of keratinocyte growth factor down-regulates the pulmonary capacity of acetylcholine production.

Keratinocyte growth factor protects the lung against various injurious stimuli. The protective mechanisms, however, are not yet fully understood. The aim of this study is to determine the influence of keratinocyte growth factor on the pulmonary capacity to synthesize acetylcholine, a potent regulator of pulmonary functions which is potentially involved in lung damage. Rats were treated twice (days 1 and 2) intratracheally with keratinocyte growth factor and analyzed at day 4. The mRNA expression of choline acetyltransferase - the acetylcholine synthesizing enzyme - was analyzed by real-time RT-PCR in the lung and in isolated alveolar epithelial type II cells. Choline acetyltransferase protein was assessed by immunoblotting and immunohistochemistry. Finally, pulmonary acetylcholine content was assessed biochemically. Keratinocyte growth factor-treatment led to decreased levels of choline acetyltransferase mRNA in the lung and in isolated alveolar epithelial type II cells. Accordingly, pulmonary choline acetyltransferase protein levels were reduced and pulmonary acetylcholine content declined from 2.8 nmol (control) to 0.4 nmol acetylcholine per gram of wet weight. In conclusion, the present data show that the potent regulator of pulmonary functions, acetylcholine, is produced by the major pulmonary target cell of keratinocyte growth factor, that is alveolar epithelial type II cells. Acetylcholine synthesis is down-regulated by keratinocyte growth factor administration which might contribute to lung protection and to harmonize surfactant homeostasis under conditions of keratinocyte growth factor-induced alveolar epithelial type II cell hyperplasia.