Two novel α7 nicotinic acetylcholine receptor ligands: in vitro properties and their efficacy in collagen-induced arthritis in mice.

INTRODUCTION: The cholinergic anti-inflammatory pathway can downregulate inflammation via the release of acetylcholine (ACh) by the vagus nerve. This neurotransmitter binds to the α7 subunit of nicotinic acetylcholine receptors (α7nAChR), expressed on macrophages and other immune cells. We tested the pharmacological and functional profile of two novel compounds, PMP-311 and PMP-072 and investigated their role in modulating collagen-induced arthritis (CIA) in mice. METHODS: Both compounds were characterized with binding, electrophysiological, and pharmacokinetic studies. For in vivo efficacy studies in the CIA model the compounds were administered daily by oral gavage from day 20 till sacrifice at day 34. Disease progression was monitored by visual clinical scoring and measurement of paw swelling. Inflammation and joint destruction were examined by histology and radiology. RESULTS: Treatment with PMP-311 was effective in preventing disease onset, reducing clinical signs of arthritis, and reducing synovial inflammation and bone destruction. PMP-072 also showed a trend in arthritis reduction at all concentrations tested. The data showed that while both compounds bind to α7nAChR with high affinity, PMP-311 acts like a classical agonist of ion channel activity, and PMP-072 can actually act as an ion channel antagonist. Moreover, PMP-072 was clearly distinct from typical competitive antagonists, since it was able to act as a silent agonist. It synergizes with the allosteric modulator PNU-120596, and subsequently activates desensitized α7nAChR. However, PMP-072 was less efficacious than PMP-311 at both channel activation and desensitization, suggesting that both conducting and non-conducting states maybe of importance in driving an anti-inflammatory response. Finally, we found that the anti-arthritic effect can be observed despite limited penetration of the central nervous system. CONCLUSIONS: These data provide direct evidence that the α7nAChR in immune cells does not require typical ion channel activation to exert its antiinflammatory effects.

Role of the cholinergic nervous system in rheumatoid arthritis: aggravation of arthritis in nicotinic acetylcholine receptor α7 subunit gene knockout mice.

BACKGROUND: The alpha7 subunit of nicotinic acetylcholine receptors (alpha7nAChR) can negatively regulate the synthesis and release of proinflammatory cytokines by macrophages and fibroblast-like synoviocytes in vitro. In addition, stimulation of the alpha7nAChR can reduce the severity of arthritis in murine collagen-induced arthritis (CIA). OBJECTIVE: To provide more insight into the role of the alpha7nAChR in the pathogenesis of arthritis by investigating the effect of the absence of alpha7nAChR in CIA in alpha7-deficient (alpha7nAChR(-/-)) compared with wild-type (WT) mice. METHODS: CIA was induced in alpha7nAChR(-/-) and WT littermate mice at day 0 by immunisation with chicken collagen type II (cCII) followed by a booster injection with cCII on day 20. Mice were killed on day 44 or day 63 and arthritis activity as well as radiological and histological damage were scored. The effects on the immune response were evaluated by measurement of antigen-specific antibodies and cytokines, and evaluation of the effects on antigen-specific stimulated spleen cells. RESULTS: In alpha7nAChR(-/-) mice a significant increase in the incidence and severity of arthritis as well as increased synovial inflammation and joint destruction were seen. Exacerbation of CIA was associated with elevated systemic proinflammatory cytokines and enhanced T-helper cell 1 (Th1)-cytokine and tumour necrosis factor alpha production by spleen cells. Moreover, a specific decrease in the collagen-specific 'Th1-associated' IgG2a response was seen, whereas IgG1 titres were unaffected. CONCLUSIONS: The results presented here indicate that immune cell function in a model of rheumatoid arthritis is regulated by the cholinergic system and, at least in part, mediated by the alpha7nAChR.

Stimulation of nicotinic acetylcholine receptors attenuates collagen-induced arthritis in mice.

OBJECTIVE: The parasympathetic nervous system, through the vagus nerve, can down-regulate inflammation in vivo by decreasing the release of cytokines, including tumor necrosis factor alpha (TNFalpha), by activated macrophages. The vagus nerve may exert antiinflammatory actions via a specific effect of its principal neurotransmitter, acetylcholine, on the alpha7 subunit of nicotinic acetylcholine receptors (alpha7nAChR) on macrophages. The present study was undertaken to obtain insight into the role of the cholinergic antiinflammatory pathway in arthritis. METHODS: To inhibit the cholinergic antiinflammatory pathway, mice were subjected to unilateral cervical vagotomy or sham surgery, after which arthritis was induced with type II collagen. In a separate study, nicotine was added to the drinking water of mice with collagen-induced arthritis (CIA). In addition, we investigated the effects of intraperitoneally (IP)-injected nicotine and the specific alpha7nAChR agonist AR-R17779. RESULTS: Clinical arthritis was exacerbated by vagotomy and ameliorated by oral nicotine administration. Moreover, oral nicotine inhibited bone degradation and reduced TNFalpha expression in synovial tissue. Both IP-injected nicotine and AR-R17779 ameliorated clinical arthritis and reduced synovial inflammation. This was accompanied by a reduction of TNFalpha levels in both plasma and synovial tissue. The effect of AR-R17779 was more potent compared with that of nicotine and was associated with delayed onset of the disease as well as with protection against joint destruction. CONCLUSION: These data provide the first evidence of a role of the cholinergic antiinflammatory pathway in the murine CIA model of rheumatoid arthritis.

Activation of the cholinergic anti-inflammatory pathway ameliorates postoperative ileus in mice.

BACKGROUND & AIMS: We previously showed that intestinal inflammation is reduced by electrical stimulation of the efferent vagus nerve, which prevents postoperative ileus in mice. We propose that this cholinergic anti-inflammatory pathway is mediated via alpha7 nicotinic acetylcholine receptors expressed on macrophages. The aim of this study was to evaluate pharmacologic activation of the cholinergic anti-inflammatory pathway in a mouse model for postoperative ileus using the alpha7 nicotinic acetylcholine receptor-agonist AR-R17779. METHODS: Mice were pretreated with vehicle, nicotine, or AR-R17779 20 minutes before a laparotomy (L) or intestinal manipulation (IM). Twenty-four hours thereafter gastric emptying was determined using scintigraphy and intestinal muscle inflammation was quantified. Nuclear factor-kappaB transcriptional activity and cytokine production was assayed in peritoneal macrophages. RESULTS: Twenty-four hours after surgery IM led to a delayed gastric emptying compared with L (gastric retention: L(saline) 14% +/- 4% vs IM(saline) 38% +/- 10%, P = .04). Pretreatment with AR-R17779 prevented delayed gastric emptying (IM(AR-R17779) 15% +/- 4%, P = .03). IM elicited inflammatory cell recruitment (L(saline) 50 +/- 8 vs IM(saline) 434 +/- 71 cells/mm(2), P = .001) which was reduced by AR-R17779 pretreatment (IM(AR-R17779) 231 +/- 32 cells/mm(2), P = .04). An equimolar dose of nicotine was not tolerated. Subdiaphragmal vagotomy did not affect the anti-inflammatory properties of AR-R17779. In peritoneal macrophages, both nicotinic agonists reduced nuclear factor kappaB transcriptional activity and proinflammatory cytokine production, with nicotine being more effective than AR-R17779. CONCLUSIONS: AR-R17779 treatment potently prevents postoperative ileus, whereas toxicity limits nicotine administration to ineffective doses. Our data further imply that nicotinic inhibition of macrophage activation may involve other receptors in addition to alpha7 nicotinic acetylcholine receptor.

Local stimulation of alpha7 cholinergic receptors inhibits LPS-induced TNF-alpha release in the mouse lung.

The cholinergic nervous system can inhibit the release of proinflammatory cytokines such as TNF-alpha from LPS-stimulated macrophages. Acetylcholine, the principal neurotransmitter of the vagus nerve, is the key mediator of this so-called cholinergic anti-inflammatory pathway, specifically interacting with alpha7 cholinergic receptors expressed by macrophages and other cell types to inhibit TNF-alpha production. The aim of the current study was to determine the capacity of the selective alpha7 cholinergic receptor agonist 3-(2,4-dimethoxybenzylidene) anabaseine (GTS-21), administered locally into the airways, to inhibit LPS-induced inflammatory responses in the mouse lung in vivo. GTS-21 dose-dependently inhibited LPS-induced TNF-alpha release by MH-S mouse alveolar macrophages in vitro. Intranasal inoculation with GTS-21 also dose-dependently inhibited TNF-alpha release into the lung compartment after intrapulmonary delivery of LPS in mice in vivo, whereas IL-6 concentrations were not affected. However, GTS-21 did not influence the influx of neutrophils into bronchoalveolar lavage fluid elicited by LPS and increased the concentrations of the neutrophil-attracting chemokines cytokine-induced neutrophil chemoattractant and macrophage inflammatory protein 2. These data indicate that local administration of GTS-21 inhibits TNF-alpha release in the lung during LPS-induced inflammation.

Intrapulmonary delivery of ethyl pyruvate attenuates lipopolysaccharide- and lipoteichoic acid-induced lung inflammation in vivo.

Ethyl pyruvate (EP) is a stable pyruvate derivative that has been shown to exert anti-inflammatory effects in various models of systemic inflammation including endotoxemia. We here sought to determine the local effects of EP, after intrapulmonary delivery, in models of lung inflammation induced by instillation via the airways of either lipopolysaccharide (LPS, a constituent of the gram-negative bacterial cell wall) or lipoteichoic acid (LTA, a component of the gram-positive bacterial cell wall). For this, we first established that EP dose dependently reduced the responsiveness of mouse MH-S alveolar macrophages and mouse MLE-15 and MLE-12 respiratory epithelial cells to stimulation with LPS or LTA in vitro. We then showed that intranasal administration of EP dose dependently inhibited tumor necrosis factor alpha release in bronchoalveolar lavage fluid of mice challenged with either LPS or LTA via the airways. Moreover, EP reduced the recruitment of neutrophils into the bronchoalveolar space after either LPS or LTA administration. These data suggest that intrapulmonary delivery of EP diminishes lung inflammation induced by LPS or LTA, at least in part by targeting alveolar macrophages and respiratory epithelial cells.

Stimulation of alpha 7 cholinergic receptors inhibits lipopolysaccharide-induced neutrophil recruitment by a tumor necrosis factor alpha-independent mechanism.

The cholinergic nervous system controls inflammation by inhibiting the release of proinflammatory cytokines such as tumor necrosis factor (TNF) alpha from lipopolysaccharide (LPS)-stimulated macrophages. The key endogenous mediator of this so-called cholinergic anti-inflammatory pathway is acetylcholine, the principal neurotransmitter of the vagus nerve, which specifically interacts with alpha7 cholinergic receptors expressed by macrophages and other cell types to inhibit TNF-alpha production. We here investigated the capacity of the selective alpha7 cholinergic receptor agonist 3-(2,4-dimethoxybenzylidene) anabaseine (GTS-21) to inhibit LPS-induced inflammatory responses in mice in vivo. To this end, mice received an intraperitoneal injection of LPS (from Escherichia coli, 200 microg) preceded by GTS-21 (4 mg/kg) or vehicle. GTS-21 strongly inhibited LPS-induced TNF-alpha release into the peritoneal cavity and the circulation. In addition, GTS-21 attenuated the influx of neutrophils into peritoneal fluid upon administration of LPS. This inhibitory effect on neutrophil recruitment by GTS-21 was independent of its effect on TNF-alpha release, considering that etanercept, a potent TNF-alpha-blocking protein containing the extracellular domain of the p75 TNF-alpha receptor, did not influence LPS-induced neutrophil influx either in the presence or in the absence of GTS-21 treatment. GTS-21 did not reduce the local secretion of macrophage inflammatory protein 2 and keratinocyte-derived cytokine, suggesting that altered concentrations of these neutrophil-attracting chemokines did not contribute to GTS-21-induced inhibition of neutrophil migration. These data identify a novel anti-inflammatory effect of chemical alpha7 cholinergic receptor stimulation that is independent from its capacity to inhibit TNF-alpha production.

Selective alpha7-nicotinic acetylcholine receptor agonist GTS-21 improves survival in murine endotoxemia and severe sepsis.

OBJECTIVE: Tumor necrosis factor and high mobility group box 1 are critical cytokine mediators of inflammation. The efferent vagus nerve inhibits cytokine release through alpha7-nicotinic acetylcholine receptor-mediated cholinergic signaling. Here we studied whether GTS-21, a selective alpha7-nicotinic acetylcholine receptor agonist, inhibits proinflammatory cytokines in vitro and in vivo and improves survival in murine endotoxemia and severe sepsis. DESIGN: Randomized and controlled in vitro and in vivo study. SETTINGS: Research laboratory and animal facility rooms. SUBJECTS: RAW 264.7 cells and BALB/c mice treated with endotoxin or subjected to cecal ligation and puncture (CLP). INTERVENTIONS: RAW 264.7 cells were exposed to endotoxin (4 ng/mL or 10 ng/mL) in the presence or absence of GTS-21 (1-100 muM), and tumor necrosis factor and high mobility group box 1 release and nuclear factor-kappaB activation were analyzed. Mice were treated with GTS-21 (0.4 mg/kg or 4 mg/kg, intraperitoneally) or saline 30 mins before endotoxin (6 mg/kg, intraperitoneally), and serum tumor necrosis factor was analyzed 1.5 hrs after the onset of endotoxemia. In survival experiments, mice were treated with GTS-21 (0.4 or 4.0 mg/kg, intraperitoneally) or saline 30 mins before and 6 hrs after endotoxin and then twice daily for 3 days. Severe sepsis was induced by CLP. Mice were treated with GTS-21 (4 mg/kg) or saline immediately and 6 hrs and 24 hrs after CLP, and serum high mobility group box 1 was analyzed 30 hrs after CLP. In survival experiments, GTS-21 (0.4 or 4 mg/kg) treatment was initiated 24 hrs after CLP and continued twice daily for 3 days. MEASUREMENTS AND MAIN RESULTS: GTS-21 dose-dependently inhibited tumor necrosis factor and high mobility group box 1 release and nuclear factor-kappaB activation in vitro. GTS-21 (4 mg/kg) significantly inhibited serum tumor necrosis factor during endotoxemia and improved survival (p < .0001). GTS-21 (4 mg/kg) significantly inhibited serum high mobility group box 1 levels in CLP mice and improved survival (p < .0006). CONCLUSION: These findings are of interest for the development of alpha7-nicotinic acetylcholine receptor agonists as a new class of anti-inflammatory therapeutics.

The vagus nerve and nicotinic receptors modulate experimental pancreatitis severity in mice.

BACKGROUND & AIMS: The nervous system, through the vagus nerve, controls inflammation by decreasing the release of tumor necrosis factor-alpha from endotoxin stimulated macrophages. This anti-inflammatory effect is mediated by an interaction of acetylcholine, the principal neurotransmitter of the vagus nerve, with macrophage cholinergic nicotinic receptors expressing the alpha7 subunit. METHODS: To determine the role of this "nicotinic anti-inflammatory pathway" in experimental pancreatitis, we induced pancreatitis in mice by 12 hourly intraperitoneal injections of cerulein. Pancreatitis was preceded by unilateral left cervical vagotomy or pretreatment with the nicotinic receptor antagonist mecamylamine or by pretreatment with the selective alpha7 nicotinic receptor agonist 3-(2,4-dimethoxybenzylidene) anabaseine (GTS-21). RESULTS: Vagotomy or pretreatment with mecamylamine resulted in an enhanced severity of pancreatitis, as reflected by histology, edema, plasma hydrolases, and interleukin-6 levels. Furthermore, the number of neutrophils migrated to the pancreas was increased in these mice, as shown by myeloperoxidase content and intrapancreatic staining of neutrophils. Conversely, GTS-21 pretreatment strongly decreased the severity of pancreatitis. Pancreatitis-associated pulmonary inflammation was independent of the integrity of the vagus nerve and nicotinic receptors. CONCLUSIONS: This study provides the first evidence for a therapeutic potential of the vagus nerve and the "nicotinic anti-inflammatory pathway" in attenuating inflammation and injury during experimental pancreatitis.