Elsevier

Biochemical Pharmacology

Volume 78, Issue 7, 1 October 2009, Pages 863-872
Biochemical Pharmacology

GTS-21 inhibits pro-inflammatory cytokine release independent of the Toll-like receptor stimulated via a transcriptional mechanism involving JAK2 activation

https://doi.org/10.1016/j.bcp.2009.06.096Get rights and content

Abstract

The vagus nerve can limit inflammation via the α7 nicotinic acetylcholine receptor (α7nAChR). Selective pharmacological stimulation of the α7nAChR may have therapeutic potential for the treatment of inflammatory conditions. We determined the anti-inflammatory potential of GTS-21, an α7nAChR-selective partial agonist, on primary human leukocytes and compared it with nicotine, the nAChR agonist widely used for research into the anti-inflammatory effects of α7nAChR stimulation. Furthermore, we investigated whether the effects of both nicotinic agonists were restricted to specific Toll-like receptors (TLRs) stimulated and explored the mechanism behind the anti-inflammatory effect of GTS-21.

GTS-21 and nicotine inhibited the release of pro-inflammatory cytokines in peripheral blood mononuclear cells (PBMCs), monocytes and whole blood independent of the TLR stimulated, with higher potency/efficacy for GTS-21 compared to nicotine. The anti-inflammatory cytokine IL-10 was relatively unaffected by both nicotinic agonists. The effects of GTS-21 and nicotine could not be reversed by nAChR antagonists, while the JAK2 inhibitor AG490 abolished the anti-inflammatory effects. GTS-21 downregulated monocyte cell-surface expression of TLR2, TLR4 and CD14. qPCR analysis demonstrated that the anti-inflammatory effect of GTS-21 is mediated at the transcriptional level and involves JAK2-STAT3 activation.

In conclusion, GTS-21 has a profound anti-inflammatory effect in human leukocytes and that GTS-21 is more potent/efficacious than nicotine. The absence of a blocking effect of nAChR antagonists in human leukocytes might indicate different pharmacological properties of the α7nAChR in human leukocytes compared to other cell types. GTS-21 may be promising from a therapeutic perspective because of its suitability for human use.

Introduction

In the past few years, a novel link between the vagus nerve and the inflammatory responses has been established. In addition to “sensing” focal inflammation in the periphery and relaying it to the brain via afferent fibers [1], [2], [3], recent work has demonstrated that the efferent vagus nerve can modulate the inflammatory response in a reflex-like fashion, termed “the cholinergic anti-inflammatory pathway”[4]. It has become clear that the α7 nicotinic acetylcholine receptor (α7nAChR), expressed in various cell types including human leukocytes [5], is an essential regulator of this anti-inflammatory effect of the vagus nerve [6]. Consequently, more specific agonists of this receptor were identified or developed and used in various studies [7], [8], [9]. To date, one of the most effective α7-selective partial agonists for modulating inflammatory responses is 3-(2,4-dimethoxybenzylidene)-anabaseine (GTS-21) which has proven to be effective in attenuating the immune response and improving outcome in animal models of pancreatitis [9], endotoxemia [8], [7], sepsis [7], acute lung injury [10], [11] and ischemia-reperfusion injury [12]. However, its anti-inflammatory potential in human inflammatory cells has never been evaluated. This is of particular interest because GTS-21, which has been primarily developed for the treatment of Alzheimer's disease, has been administered to human volunteers and patients and is well tolerated without clinically significant safety findings [13]. Therefore, GTS-21 may have therapeutic potential for the treatment of inflammatory conditions and is preferred above nicotine which lacks pharmacologic specificity and has toxic side effects and the potential to produce physical dependence (addiction). Although comparisons between GTS-21 and non-selective nAChR agonists have been reported at α7nAChR receptor activation level in electrophysiological studies using heterologous expression in Xenopus laevis oocytes [14], [15], the immunomodulating effects of selective and non-selective agonists of the α7nAChR have not been compared. Furthermore, in vitro studies investigating the cholinergic anti-inflammatory pathway almost exclusively used the principal Toll-like receptor 4 (TLR4) agonist LPS as a trigger for inflammation. It is unknown whether the cholinergic anti-inflammatory pathway is restricted to certain TLRs stimulated or is a general mechanism not constrained to a specific stimulus.

Finally, the mechanism by which α7nAChR stimulation attenuates pro-inflammatory cytokine production has not been fully elucidated and sparcely studied in human cells. A role for the JAK2-STAT3 pathway [16], [17], [18], [19] as well as suppression of NFκB transcriptional activity [20], [21] is implied. However, the cholinergic anti-inflammatory pathway is believed to be regulated at a post-transcriptional level [4], [16]. Mechanistic studies regarding the anti-inflammatory effect of GTS-21 are limited to one study reporting decreased NFκB activity in a murine cell line [7].

In this study, we investigated for the first time the anti-inflammatory potential of GTS-21 on primary human leukocytes and compared it with nicotine. Furthermore, we investigated whether the effects of both nicotinic agonists were restricted to specific TLRs stimulated and whether they affected cell-surface expression of receptors involved in the innate immune response. Finally, we studied whether the anti-inflammatory effects of GTS-21 and nicotine are regulated at the transcriptional level and determined the involvement of the JAK-STAT signal transduction pathway.

Section snippets

General reagents

RPMI culture medium (RPMI 1640 Dutch modification, ICN Biomedicals; Costa Mesa, CA, USA) was supplemented with gentamicin 10 μg/mL, l-glutamine 10 mM and pyruvate 10 mM. GTS-21 was obtained from the University of Florida (a kind gift of Prof. Dr. Roger L. Papke) and from Comentis Inc. (South San Francisco, CA, USA). No differences in potency/efficacy between GTS-21 from the University of Florida and from Comentis Inc. were observed (data not shown). Nicotine (liquid, naturally occurring isomer),

Nicotine and GTS-21 dose-dependently inhibit LPS-induced cytokine production in PBMCs

To determine the effect of nAChR agonists on LPS-induced cytokine release we incubated human PBMCs with 1 ng/mL LPS in combination with nicotine or GTS-21 for 22 h. The classic nAChR agonist nicotine dose-dependently inhibited production of the pro-inflammatory cytokines TNF- α, IL-1β and IL-6 (Fig. 1) with a maximum inhibition of 30 ± 4%, 65 ± 2% and 36 ± 5%, respectively at the highest dose of nicotine used (1 mM). The anti-inflammatory cytokine IL-10 was relatively unaffected, with a small but

Discussion

The cholinergic anti-inflammatory pathway may represent new treatment options for inflammatory conditions such as sepsis, acute lung injury and autoimmune diseases. Vagus nerve stimulation in humans is a very invasive procedure and is not feasible in acute situations. Therefore, pharmacological stimulation of the cholinergic anti-inflammatory pathway via the α7nAChR is a more practical approach. The non-specific nAChR agonist nicotine has little therapeutic potential because of its toxicity and

Acknowledgements

The authors would kindly like to thank Prof. Dr. Roger L. Papke of the University of Florida and Comentis Inc. for supplying us with GTS-21 and Trees Jansen for help with the Luminex cytokine measurements. M. Kox and J.C. Pompe are recipients of a grant of the Radboud University Nijmegen Medical Centre Trauma Section/Intensive Care Medicine.

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