Cholinergic signaling controls immune functions and promotes homeostasis.

Acetylcholine (ACh) was created by nature as one of the first signaling molecules, expressed already in procaryotes. Based on the positively charged nitrogen, ACh could initially mediate signaling in the absence of receptors. When evolution established more and more complex organisms the new emerging organs systems, like the smooth and skeletal muscle systems, energy-generating systems, sexual reproductive system, immune system and the nervous system have further optimized the cholinergic signaling machinery. Thus, it is not surprising that ACh and the cholinergic system are expressed in the vast majority of cells. Consequently, multiple common interfaces exist, for example, between the nervous and the immune system. Research of the last 20 years has unmasked these multiple regulating mechanisms mediated by cholinergic signaling and thus, the biological role of ACh has been revised. The present article summarizes new findings and describes the role of both non-neuronal and neuronal ACh in protecting the organism from external and internal health threats, in providing energy for the whole organism and for the individual cell, controling immune functions to prevent inflammatory dysbalance, and finally, the involvement in critical brain functions, such as learning and memory. All these capacities of ACh enable the organism to attain and maintain homeostasis under changing external conditions. However, the existence of identical interfaces between all these different organ systems complicates the research for new therapeutic interventions, making it essential that every effort should be undertaken to find out more specific targets to modulate cholinergic signaling in different diseases.

Immunomodulatory role of non-neuronal cholinergic signaling in myocardial injury.

Whereas prior studies have demonstrated an important immunomodulatory role for the neuronal cholinergic system in the heart, the role of the non-neuronal cholinergic system is not well understood. To address the immunomodulatory role of the non-neuronal cholinergic system in the heart we used a previously validated diphtheria toxin (DT)-induced cardiomyocyte ablation model (Rosa26-DTMlc2v-Cre mice). DT-injected Rosa26-DTMlc2v-Cre mice were treated with diluent or Pyridostigmine Bromide (PYR), a reversible cholinesterase inhibitor. PYR treatment resulted in increased survival and decreased numbers of MHC-IIlowCCR2+ macrophages in DT-injected Rosa26-DTMlc2v-Cre mice compared to diluent treated Rosa26-DTMlc2v-Cre mice. Importantly, the expression of CCL2/7 mRNA and protein was reduced in the hearts of PYR-treated mice. Backcrossing Rosa26-DTMlc2v-Cre mice with a transgenic mouse line (Chat-ChR2) that constitutively overexpresses the vesicular acetylcholine transporter (VAChT) resulted in decreased expression of Ccl2/7 mRNA and decreased numbers of CD68+ cells in DT-injured Rosa26-DTMlc2v-Cre/Chat-ChR2 mouse hearts, consistent with the pharmacologic studies with PYR. In vitro studies with cultures of LPS-stimulated peritoneal macrophages revealed a concentration-dependent reduction in CCL2 secretion following stimulation with ACh, nicotine and muscarine. Viewed together, these findings reveal a previously unappreciated immunomodulatory role for the non-neuronal cholinergic system in regulating homeostatic responses in the heart following tissue injury.

Vagal-immune interactions involved in cholinergic anti-inflammatory pathway.

Inflammation and other immune responses are involved in the variety of diseases and disorders. The acute response to endotoxemia includes activation of innate immune mechanisms as well as changes in autonomic nervous activity. The autonomic nervous system and the inflammatory response are intimately linked and sympathetic and vagal nerves are thought to have anti-inflammation functions. The basic functional circuit between vagus nerve and inflammatory response was identified and the neuroimmunomodulation loop was called cholinergic anti-inflammatory pathway. Unique function of vagus nerve in the anti-inflammatory reflex arc was found in many experimental and pre-clinical studies. They brought evidence on the cholinergic signaling interacting with systemic and local inflammation, particularly suppressing immune cells function. Pharmacological/electrical modulation of vagal activity suppressed TNF-alpha and other proinflammatory cytokines production and had beneficial therapeutic effects. Many questions related to mapping, linking and targeting of vagal-immune interactions have been elucidated and brought understanding of its basic physiology and provided the initial support for development of Tracey´s inflammatory reflex. This review summarizes and critically assesses the current knowledge defining cholinergic anti-inflammatory pathway with main focus on studies employing an experimental approach and emphasizes the potential of modulation of vagally-mediated anti-inflammatory pathway in the treatment strategies.

Aflatoxins produced by Aspergillus parasiticus present in the diet of quails increase the activities of cholinesterase and adenosine deaminase.

The aim of this study was to evaluate the effects of aflatoxins on cholinesterases (acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and adenosine deaminase (ADA) activities in quails. For this, twenty male quails were randomly distributed into two groups (n = 10 each): the group A was composed by quails that received feed without aflatoxin (the control group); while the group B was composed by quails that received feed contaminated with 200 ppm/kg of feed of aflatoxin. On day 20, the animals were euthanized to measure the activities of AChE (total blood and brain), BChE (serum) and ADA (serum, liver, and brain), as well as for histopathological analyses (liver and intestine). AChE, BChE, and ADA levels increased in animals intoxicated by aflatoxin compared to the control group. The presence of aflatoxin lead to severe hydropic degeneration of hepatocytes and small focus of hepatocyte necrosis. In conclusion, aflatoxins poisoning increased AChE, BChE, and ADA activities, suggesting the involvement of these enzymes during this type of intoxication, in addition to the fact that they are well known molecules that participate in physiological and pathological events as inflammatory mediators. In summary, increased AChE, BChE and ADA activities contribute directly to the inflammatory process and tissue damage, and they might be involved in disease development.

The intestinal cholinergic anti-inflammatory pathway.

The main task of the immune system is to distinguish and respond accordingly to 'danger' or 'non-danger' signals. This is of critical importance in the gastrointestinal tract in which immune cells are constantly in contact with food antigens, symbiotic microflora and potential pathogens. This complex mixture of food antigens and symbionts are essential for providing vital nutrients, so they must be tolerated by the intestinal immune system to prevent aberrant inflammation. Therefore, in the gut the balance between immune activation and tolerance should be tightly regulated to maintain intestinal homeostasis and to prevent hypersensitivity to harmless luminal antigens. Loss of this delicate equilibrium can lead to abnormal activation of the intestinal immune system resulting in devastating gastrointestinal disorders such as inflammatory bowel disease (IBD). Recent evidence supports the idea that the central nervous system interacts dynamically via the vagus nerve with the intestinal immune system to modulate inflammation through humoral and neural pathways, using a mechanism also referred to as the intestinal cholinergic anti-inflammatory pathway. In this review, we will focus on the current understanding of the mechanisms and neuronal circuits involved in the intestinal cholinergic anti-inflammatory pathway. Further investigation on the crosstalk between the nervous and intestinal immune system will hopefully provide new insights leading to the identification of innovative therapeutic approaches to treat intestinal inflammatory diseases.

The interface between cholinergic pathways and the immune system and its relevance to arthritis.

The nervous and immune systems are likely to be interacting in arthritis, with the possible involvement of both neural and non-neural cholinergic transmission. Centrally acting muscarinic agonists, electrical stimulation of the vagus and treatment with nicotinic receptor agonists can all act systemically to reduce inflammation, although the responsible pathways are incompletely understood. While this 'cholinergic anti-inflammatory pathway' is widely viewed as a significant pathophysiological mechanism controlling inflammation, the evidence supporting this view is critically reviewed and considered inconclusive; an alternative pathway via sympathetic nerves is implicated. This review also discusses how cholinergic pathways, both neural and non-neural, may impact on inflammation and specifically arthritis. Nicotinic agonists have been reported to reduce the incidence and severity of murine arthritis, albeit an observation we could not confirm, and clinical studies in rheumatoid arthritis have been proposed and/or are underway. While the therapeutic potential of nicotinic agonists and vagal stimulation is clear, we suggest that the 'cholinergic anti-inflammatory pathway' should not be uncritically embraced as a significant factor in the pathogenesis of rheumatoid arthritis.

The influence of cholinergic agents on histamine release from HMC-1 human mast cell line stimulated with IgG, C-reactive protein and compound 48/80.

AIMS: The aim of this work is to study the role of acetylcholine (ACh) receptors (AChRs) in the regulation of FcγR activity in human mast cells (MC) activated by aggregated IgG (aIgG) and CRP. MC, the key regulators at the interface of innate and acquired immunity, have abundant Fc receptors for IgG (FcγRII) which indicate the role of their ligands, IgG and C-reactive protein (CRP), in regulating MC activity. Cholinergic control of FcγR-dependent MC functions is poorly defined. MAIN METHODS: HMC-1 culture of human MC; cell incubations with cholinergic drugs and FcγR ligands such as heat aggregated human IgG or purified human CRP; compound 48/80, a known histamine liberator employing G protein-coupled receptors, was used as a positive control of MC degranulation; assessment of histamine release. KEY FINDINGS: Both nAChR and mAChR antagonists (hexamethonium and methacine, respectively), per se, elevated histamine-releasing activity of the HMC-1 and suppressed the MC responses to most of investigated activators (carbachol, compound 48/80, and to a lesser extent aIgG). Two blockers together should be applied to aIgG-stimulated cells in order to obtain appreciable suppression of histamine release. The FcγR-mediated HMC-1 cell response to CRP was the least sensitive to attenuation by ACh signaling. SIGNIFICANCE: The data obtained suggest the involvement of ACh in the functioning of other receptor systems. Our results indicate that AChRs are closely associated with G protein-coupled receptor-induced reactions of MC and optionally with FcγR-dependent functions. CONCLUSION: The data presented demonstrate that AChRs and endogenous ACh are involved in regulating mast cell degranulation and histamine release by affecting the functions of receptors to compound 48/80 and, less, FcγRs.

Role of the cholinergic antiinflammatory pathway in murine autoimmune myocarditis.

RATIONALE: This study was performed to gain insights into novel therapeutic approaches for the treatment of autoimmune myocarditis. OBJECTIVE: Chemical stimulation of the efferent arm of the vagus nerve through activation of nicotinic acetylcholine receptor subtype-7α (α7-nAChR) has been shown to be protective in several models of inflammatory diseases. In the present study, we investigated the potentially protective effect of vagus nerve stimulation on myocarditis. METHODS AND RESULTS: A/J mice were immunized with cardiac troponin I (TnI) to induce autoimmune myocarditis. Mice were exposed to drinking water that contained nicotine in different concentrations and for different time periods (for 3 days at 12.5 mg/L; 3 days at 125 mg/L; 21 days at 12.5 mg/L; and 21 days at 125 mg/L after first immunization). TnI-immunized mice with no pharmacological treatment showed extensive myocardial inflammation and fibrosis and significantly elevated levels of interleukin-6 and tumor necrosis factor-α. Furthermore, elevated levels of mRNA transcripts of proinflammatory chemokines (monocyte chemoattractant protein-1, macrophage inflammatory protein-1ß, and RANTES) and chemokine receptors (CCR1, CCR2, and CCR5) were found. Oral nicotine administration reduced inflammation within the myocardium, decreased the production of interleukin-6 and tumor necrosis factor-α, and downregulated the expression of monocyte chemoattractant protein-1, macrophage inflammatory protein-1ß, RANTES, CCR1, CCR2, and CCR5. In addition, nicotine treatment resulted in decreased expression of matrix metalloproteinase-14, natriuretic peptide precursor B, tissue inhibitor of metalloproteinase-1, and osteopontin, proteins that are commonly involved in heart failure. Finally, we found that nicotine reduced levels of pSTAT3 (phosphorylated signal transducer and activator of transcription 3) protein expression within the myocardium. Neostigmine treatment did not affect the progression of myocarditis. CONCLUSIONS: We showed that activation of the cholinergic antiinflammatory pathway with nicotine reduces inflammation in autoimmune myocarditis. Our results may open new possibilities in the therapeutic management of autoimmune myocarditis.

Cholinergic control of inflammation.

Cytokine production is necessary to protect against pathogens and promote tissue repair, but excessive cytokine release can lead to systemic inflammation, organ failure and death. Inflammatory responses are finely regulated to effectively guard from noxious stimuli. The central nervous system interacts dynamically with the immune system to modulate inflammation through humoral and neural pathways. The effect of glucocorticoids and other humoral mediators on inflammatory responses has been studied extensively in the past decades. In contrast, neural control of inflammation has only been recently described. We summarize autonomic regulation of local and systemic inflammation through the 'cholinergic anti-inflammatory pathway', a mechanism consisting of the vagus nerve and its major neurotransmitter, acetylcholine, a process dependent on the nicotinic acetylcholine receptor alpha7 subunit. We recapitulate additional sources of acetylcholine and their contribution to the inflammatory response, as well as acetylcholine regulation by acetylcholinesterase as a means to attenuate inflammation. We discuss potential therapeutic applications to treat diseases characterized by acute or chronic inflammation, including autoimmune diseases, and propose future research directions.

Controlling inflammation: the cholinergic anti-inflammatory pathway.

Innate immune responses and inflammation are regulated in part by neural mechanisms. In the present paper, we summarize experimental evidence that reveals that innate immunity and inflammation are controlled by the vagus nerve, previously known as a regulator of other vital physiological functions. Activation of vagus nerve cholinergic signalling inhibits TNF (tumour necrosis factor) and other pro-inflammatory cytokine overproduction through 'immune' alpha7 nicotinic receptor-mediated mechanisms. This efferent vagus nerve-based 'cholinergic anti-inflammatory pathway' has been elucidated as a critical regulator of inflammation in several experimental models of diseases. Our recent observations have shown that activation of central (brain) cholinergic transmission by selective muscarinic receptor ligands results in lower systemic TNF levels in rodents and indicate that the efferent vagus nerve may provide a functional brain-to-immune connection. Thus central cholinergic signalling is implicated in the activation of the cholinergic anti-inflammatory pathway. Electrical vagus nerve stimulation is clinically approved for the treatment of epilepsy and depression and current knowledge suggests that it could be utilized to control inflammation. Advances in understanding the receptor and molecular mechanisms of cholinergic anti-inflammatory signalling indicate that selective alpha7 nicotinic receptor agonists and centrally acting cholinergic enhancers can be used in the treatment of pathological conditions characterized by cytokine overproduction.

Compensatory changes in cortical cholinergic innervation in the rat following an immunotoxic lesion.

PURPOSE: To investigate the plastic capacity of the cholinergic system in a partial animal model of Alzheimer's disease. METHODS: Rats received unilateral lesions of the horizontal diagonal band of Broca (HDB) using a cholinergic-specific toxin, 192 IgG-saporin. After the appropriate survival time (2, 4, 8, 12 and 24 weeks post-lesion) rats were sacrificed and the brains were prepared for histology. Immunocytochemical and morphometric techniques were employed to quantify the cholinergic neurons surviving the lesion and to measure the density of cortical cholinergic fibers. RESULTS: Cell counts revealed on average a 60% reduction in cholinergic neurons on the lesioned side, compared to the spared side. This cell loss was permanent, that is, there was no significant change in the amount of cell loss over time. In correlation with this cell loss, cholinergic fibers in the target area, the entorhinal cortex (EC), were also reduced such that the density of acetylcholinesterase (AChE)-stained fibers on the lesioned side was 44% of the spared side. The density of cholinergic fibers in the EC increased significantly between 2 and 12 weeks post-lesion (p=0.0216) but remained stable at that level by 24 weeks after the lesion. CONCLUSIONS: Following a cholinergic-specific lesion, a compensatory mechanism is activated in the basal forebrain cholinergic system, such that surviving neurons, projecting to the same target, extend their terminals to occupy the denervated area. It remains to be investigated whether these sprouts are able to establish proper synaptic connections and make a functional recovery in this particular system.

Altered NGF protein levels in different brain areas after immunolesion.

Nerve growth factor (NGF) provides critical trophic support to the cholinergic basal forebrain neurons that express high levels of the low-affinity NGF receptor (p75NGFR) in the adult rat brain. Intraventricular injection of 192 IgG-saporin, made by coupling the monoclonal antibody to p75NGFR 192 IgG to the cytotoxin saporin, selectively destroys the p75NGFR-bearing neurons in the basal forebrain and was used here to examine the effects of selective cholinergic lesions on brain NGF protein levels. We showed that 192 IgG-saporin produced significant long-lasting elevation of NGF protein levels in the hippocampus, cortex, and olfactory bulb, with profound reductions of ChAT activities representing complete cholinergic deafferentations of these areas. NGF level was maintained in the basal forebrain, even though there was almost complete loss of p75NGFR-immunoreactive cells and significant decrease of ChAT activity. In addition, a mild glial response was observed in the basal forebrain, and most of the activated astroglia expressed NGF-like immunoreactivity there. The increases in NGF protein levels in the target areas of the basal forebrain were most likely due to loss of cholinergic basal forebrain neurons and retrograde transport of NGF from these areas. Glial-derived NGF is partially responsible for the maintained level of NGF in the basal forebrain after the loss of cholinergic neurons. The accumulation of NGF protein in the target areas may have some effects on synaptic rearrangement in denervated tissues.

Double-blind crossover study of high-dose cetirizine in cholinergic urticaria.

BACKGROUND: Cholinergic urticaria does not respond well to treatment with conventional antihistamines and is difficult to study because of its highly variable clinical expression which depends on the presence of eliciting factors. OBJECTIVE: We have therefore designed a double-blind, crossover, placebo-controlled trial, with a 3-week treatment period using either 20 mg/day of cetirizine or placebo. METHODS AND RESULTS: Presence of eliciting factors and symptoms were scored daily on a diary card by the patient, with a scale from 0 to 3 for erythema, wheals and pruritus. Statistical analysis was done on 11 evaluable patients during the last 2 weeks of each treatment period (to allow for 1 week of washout) and only for days when eliciting factors were present. Compared to placebo, cetirizine caused a statistically significant reduction of wheals (p = 0.015), erythema (p = 0.033), pruritus (p = 0.006) and all symptoms (p = 0.013). No adverse events were observed. CONCLUSION: These data show a high efficacy of cetirizine at twice its normally recommended dose which may be related to the specific antiallergic effects of this newer-generation antihistamine.

Cholinergic-specific glycoconjugates.

Cholinergic nerve terminals utilize glycoconjugates in several ways, as surface markers and as structural components of the synaptic vesicles present within them. The surface markers have been discovered immunochemically: antibodies raised against them are able specifically to sensitize the cholinergic subpopulation of mammalian brain synaptosomes to complement-mediated lysis. One such group of antigens (Chol-1) have been identified as a novel series of minor gangliosides having in common a sialylated N-acetylgalactosamine residue. These gangliosides may constitute the major gangliosides at cholinergic terminals. A second surface antigen (Chol-2) is thought to be a protein with an epitope in common with a Torpedo electric organ ganglioside. Cholinergic synaptic vesicles are rich in a proteoglycan which appears to assist in the sequestration of acetylcholine within the vesicle and to stabilize the vesicle membrane during cycles of exocytosis and recovery. It may be the cholinergic equivalent of the chromogranins.