it is an organic molecule and one of many neurotransmitters in the autonomic nervous system ( ans ) . there are two main classes of acetylcholine receptors ( achr ) : nicotinic acetylcholine receptors ( nachr ) and muscarinic acetylcholine receptors ( machr ) . the nachr is the best - characterized ligand - dependent , cation - selective channel receptor . nicotinic acetylcholine receptors are present in the autonomic ganglia and at the neuromuscular junction . in the brain , nachrs play an important role in the brain activity modulating synaptic and cellular functions , while in muscles they are involved in neural activation of muscle spasms . in vertebrate species , 17 different subunits of nachr have been identified ( 1 - 10 , 1 - 4 ,  ,  ,  ) . the subunits form a large number of homo - and heteropentameric receptors and all these combinations show specific pharmacological properties . stimulation of nachrs opens channels , induces an inflow of sodium ions and/or calcium ions as well as a potassium ions outflow ( fig . voltage - operated calcium channels are opened , leading to an additional flow of calcium ions . the 7 nicotinic acetylcholine receptor ( nachr ) consists of five 7 subunits and is called 7 homo - oligomeric nachr ( left ) . the 42 nachr is composed of two 4 subunits and three 2 subunits and thus is called 42 heteromeric nachr ( right ) . in both nachrs , subunits are arranged around a central pore or channel that opens when agents such as acetylcholine or nicotine bind to an nachr , allowing positively charged ions to flow through the channel into the cell . the 7 nachr principally allows passage of calcium ( ca ) ions , whereas the 42 nachr allows passage of both calcium and sodium ( na )   the terms  non - neuronal cholinergic system and  non - neuronal acetylcholine were first introduced in the 1970s by sastry and sadavongvivad . since that time , multiple studies have been performed to investigate the function of nach receptors in different non - neuronal tissues . nicotinic acetylcholine receptors were found on keratinocytes , blood cells including lymphocytes , macrophages , mast cells , dendritic cells , basophils , microglia , endothelial cells , epithelial cells and fibroblasts [ 10 , 11 ] . the expression of this receptor was also observed in tumor cells , suggesting its potential role in cancer progression . - neuronal cholinergic system was associated with changes in proliferation , differentiation , migration , adhesion , cell contact , apoptosis , and angiogenesis process [ 7 , 13 , 14 ] . the expression of nachrs was also detected in spermatogonia , spermatocytes , seminiferous tubular and sertoli cells . this suggests that the presence of the non - neuronal cholinergic system in testicular parenchyma plays a potentially important role in the differentiation of germ cells . studies performed in the past years have indicated that cholinergic transmission may modulate various aspects of both innate and adaptive immune response . it has been shown that acetylcholine  an element of the non - neuronal cholinergic system  exhibits an anti - inflammatory action . acetylcholine may affect the immune system in two different ways  autocrine / paracrine ( immune cells have the capacity of acetylcholine secretion ) or nerve stimulation [ 17 , 18 ] . cholinergic anti - inflammatory pathway response plays a role in the immune response and inflammatory cascade . the non - neuronal cholinergic system affects also immune cell proliferation , t helper differentiation , antigen presentation and cytokine production . the -7 nicotinic acetylcholine receptor ( 7-nachr ) , a part of the non - neuronal cholinergic system , plays a specific role in the immune response . it is localized in various immune cells , i.e. macrophages , dendritic cells , t and b lymphocytes , mast cells , or basophils [ 14 , 1921 ] . the cytokine suppressing mechanism of the inflammatory reflex   cholinergic anti - inflammatory pathway   requires the -7 subunit of nicotinic acetylcholine receptor ( 7-nachr ) . the release of acetylcholine in spleen suppresses production of pro - inflammatory cytokines ( e.g. tnf- ) and attenuates inflammation . signaling through the efferent arc of the inflammatory reflex regulates the innate immune response . interestingly , olofsson et al .   have demonstrated that deficiency of 7-nachrs in the bone marrow cells significantly impaired nerve regulation of tnf release , but 7-nachr deficiency in neurons , or other cells had no significant effect . authors suggested that the expression of 7-nachr on most of the immune cells , but not in the neurons or t cells , is necessary to sustain the inflammatory reflex function . furthermore , deficiency or impairment of 7-nachr signaling leads to overproduction of cytokines , and enhances tissue damage . positive agonists of 7-nachr were used in preventing tissue damage caused by the inflammatory process . sun et al .   have shown that administration of pnu-120596 , a type - ii positive allosteric modulator ( pam - ii ) of 7 nicotinic acetylcholine receptors , reduces brain injury and improves the neurological function after focal cerebral ischemia in rats . additionally , pnu-120596 administration had anti - nociceptive effects in mice models of persistent pain . macrophages isolated from the wild type mouse produced significantly lower levels of tumor necrosis factor  ( tnf- ) after lps stimulation if 5 - 10 minutes before they were pre - incubated with nicotine ( 1 , 10 m ) or acetylcholine ( 10 m ) than macrophages isolated from 7-nachr knockout mice . in lps - stimulated human macrophage cultures , acetylcholine attenuated release of cytokines ( tnf- ) , il-1 , il-6 and il-18 , but not the anti - inflammatory cytokine il-10 . have shown that an 7-nachr agonist ( gts-21 ) suppresses pro - inflammatory cytokine production ( mainly tnf- and il-1 ) in response to various toll - like receptor ( tlr ) ligands e.g. tlr2 , tlr3 , and tlr9 agonists . this relationship was also observed in culture of microglia cells where acetylcholine and nicotine pre - treatment inhibited lipopolysaccharide ( lps)-induced tnf- release . t cells possess all cholinergic elements of the non - neuronal cholinergic system : acetylcholine , choline acetyltransferase ( chat ) , muscarinic and nicotinic acetylcholine receptors and acetylcholinesterase . ach or nicotine treatment of t and b cells causes rapid and transient intercellular calcium ion signaling in these cells . demonstrated the role of the vagus nerve and ach in controlling the innate immune response ( reduction of inflammation ) . the vagus nerve stimulated acetylcholine - producing memory phenotype t cells , which were required for inhibition of cytokine production . permanent nicotine stimulation down - regulates the nachr expression and suppresses t cell activity [ 3234 ] . moreover , fujii et al .   showed that stimulation of the t cell receptor ( tcr ) using a t cell activator  phytohemagglutinin ( pha ) , induces ach synthesis and release in molt-3 and hsb-2 cells ( human leukemic t cell lines ) . it is well known that antagonists of the 7-nachr , such as -bungarotoxin ( -btx ) and methyllycaconitine ( mla ) , enhance t cell proliferation . recent data indicate that 7-nachr seems to be a key regulator of immunosuppressive functions of cd4 cd25 regulatory t cells . additionally , nordman et al .   revealed that not only 7-nachrs but also 4-nachr nicotinic receptors are involved in cd4 t - cell proliferation and helper t - cell immune response . studies performed in vitro in human dendritic cells after nicotine administration showed a decrease in their phagocytic activity and secretion of pro - inflammatory cytokines . nicotine inhibits il-12 release by lps - matured human competent dendritic cells and reduces secretion of ifn- by the primed t cells . simultaneously , nicotine administered to mice stimulated with prophylactic and therapeutic vaccines , adversely affects dendritic cells and prevents development and maintaining of ag - specific effector memory th1 cells and ab production . studies performed on rats revealed that acetylcholine , choline and nicotine induce mast cell degranulation . mishra et al .   have demonstrated that nicotine affects the late , but not the early , degranulation phase of the mast cell activation . authors suggested that nachrs localized on mast cells might be an important component for new therapeutic approaches to control allergic diseases and allergic asthma . the 7-nachr specific agonist gts-21 also shows anti - inflammatory effects on mucosal - type murine bone marrow - derived mast cells ( mbmmcs ) and inhibits ige - induced degranulation . nicotinic acetylcholine receptor also regulates the expression of cell surface proteins associated with regulation of leukocyte trafficking to peripheral inflammatory sites . cd11b on the surface of neutrophils is necessary for 2-integrin regulated neutrophil adhesion to the endothelium and neutrophil transmigration to sites of microbial infection . it has been shown that nicotine administration and vagus nerve stimulation ( vns ) significantly reduced both lps- and fmlp-(n - formyl - methionyl - leucyl - phenylalanine ) induced neutrophil surface cd11b expression via suppression of f - actin polymerization . the ach esterase plays an important role in the acetylcholine stimulation of non - neural immune cells . the enzyme is a member of the / hydrolase family and it degrades the excitatory neurotransmitter acetylcholine ( ach ) . degradation of ach by acetylcholinesterase ( ache ) is necessary for termination of the signal and may be controlled at different cell levels . ache can be incorrectly translocated from the cytoplasm to the membrane , which was observed in an autoimmune disease gilboa - geffen et al .   in non - obese diabetic mice showed that tlr9 activated by oligonucleotide bl-7040 induced an alternative nf-b activation mode resulting in a reduced peripheral ache activity . similarly , ofek et al .   observed in young volunteers an increase or decrease in the plasma ache activity 1.5 h after escherichia coli endotoxin administration compared to saline . moreover , ache function can be regulated at the expression level by alternative splicing , microrna suppression   or transcriptional control . this receptor exhibits a higher permeability for calcium ions in relation to other nachrs . as a result of increased intracellular calcium concentrations , the influx of calcium ions activates phosphorylation cascade via protein kinase c ( pkc ) , serine - threonine kinase raf-1 , mitogen - activated protein kinase ( mapk )   and/or extracellular signal - regulated kinase erk1 , erk2 . it has been shown that many intracellular proteins interact with 7-nachrs and participate in signal transduction . as a result of nachrs activation , nuclear transcription factor kappab ( nf-b ) is activated via the janus kinase 2 ( jak-2 ) , phosphatidylinositol kinases pi3k and serine - threonine protein kinase akt . other transcription factors were also activated by stimulation of nachr : activator protein-1 ( ap-1 ) , signal transducer and activator or transcription 1 ( stat1 ) , stat3 and stat5 in the jak-2/pi3k / akt pathway [ 52 , 59 ] . activated nachrs mediate carcinogenic processes including proliferation , inhibition of apoptosis , angiogenesis , migration and cell adhesion . other researchers suggest that molecular therapy strategies aimed at nachrs inhibition or sirna silencing in lung cancer may reduce tumor growth and development . stimulation of 7-nachrs in mouse macrophages in vitro and in vivo leads to an anti - inflammatory effect via the jak2stat3 signaling pathway . in turn , wang et al . have observed that activation of 7 nachrs on human macrophages inhibits activation of the nf-b signaling pathway induced by either endotoxin or tnf-. this caused inhibition of pro - inflammatory cytokines release . activation of 7-nachr , an endogenous inflammation - resolving pathway , prevents tnf- induced nf-b activation and inhibits macrophage migration into the hippocampus . a study by waldburger et al . shows that activation of 7-nachrs by an agonist inhibits cytokine production by resident fibroblast - like synoviocytes ( fls ) , which are potentially directly involved in propagation of inflammation . in another study , al - wadei et al acetylcholine , nicotine , and its nitrosated carcinogenic derivative nnk activated phosphorylation of creb ( camp response element - binding protein ) , erk , src ( proto - oncogene tyrosineprotein kinase ) and akt . also , nachrs stimulate growth and migration of pancreatic cancer cells . have shown inhibition of tnf- expression in response to lps after an 7-nachr nicotine stimulation . moreover , nicotine administration decreased the expression of primary response gene 88 protein ( myd88 ) , nf-b p65 protein , phospho - i-b , tnf- as well as nf-b activity induced by lps in airway epithelial cells ( hbe16 ) . nicotine reduced also tnf - expression through the 7 nachr / myd88/nf-b pathway . in addition , li et al .   revealed that when silencing of 1 , 5 or 7-nachrs in airway epithelial cells ( hbe16 ) with appropriate sirna , only the 7-nachr is responsible for lowering tnf- production . in normal human bronchial epithelial ( hbe ) cells , nicotine administration resulted in phosphorylation of the signaling protein p38mapk , akt   and an increase in proliferation of small cell lung cancer cell lines . it is well known that akt participates in cellular processes such as , glucose metabolism , cell cycle progression , and apoptosis . nicotine has been reported to inactivate the pro - apoptotic protein bax , also by the akt signaling pathway . in hek 273 t cells , the influx of calcium stimulated by nicotine can be blocked by -btx ( an antagonist of 7-nachr ) . activation of akt increased phosphorylation of downstream substrates including gsk-3 , p70s6k , 4ebp-1 , and fkhr and could contribute to tumorigenesis in vivo in lymphoid , breast , ovarian , prostate , and brain tissues . other study showed that in neuronal cells , 7-nachr activates the pi3k / akt pathway by activation of a src family member . nicotine at a concentration of 10 mm caused phosphorylation of mitogen - activated protein kinase p 44/42 mapk in non - small cell lung cancer ( nsclc ) cell lines . p44/42 mapk is often up - regulated in cancer and can be inhibited using nachr antagonists . moreover , nicotine can activate 7-nachrs to inhibit the expression of inducible nitric oxide synthase and nitric oxide through the mitogen - activated protein kinase ( mapk)/nf-b pathway . nicotine activation of nachr , nf-b p65 and p50 , i-b activation , and the mapk signaling pathway induced vascular endothelial cells to produce adhesion molecule : icam-1 and vcam-1 . cholinergic receptors stimulation via nachr agonists , such as nicotine and gts-21 or the vagus nerve stimulation suppresses leukocyte recruitment and the endothelial cell activation . chatterjee et al . have shown significantly reduced production of il-6-mediated monocyte chemoattractant protein-1 ( mcp-1 ) and icam-1 expression regulated through the jak2/stat3 pathway in macrovascular human umbilical vein endothelial cells ( huvecs ) and microvascular endothelial cells ( mvecs ) . authors found that cells treated with cholinergic agonists ( e.g. , nicotine and gts-21 ) significantly reduced phosphorylation of jak2 , reduced stat3 activation ( by phosphorylation and dna binding ) and decreased levels of socs3 ( suppressor of cytokine signaling ) . similarly , saeed et al . demonstrated that cholinergic agonists ( nicotine ) suppressed endothelial cell activation ( in in vitro and in vivo studies ) and leukocyte migration in a carrageenan air pouch model . on the other hand , studies performed by de jonge et al .   showed that nicotine induced stat3 activation in macrophages following lps treatment . have observed simultaneous activation of stat3 and extracellular signal regulated kinase 1/2 ( erk1/2 ) in t24 cells after nicotine administration . however , long - term nicotine exposure induced activation of stat3 and down - regulation of erk1/2 via nachr and -adrenoceptors in human bladder cancer cells ( t24 cell line ) . activation of calcium ions transport by 7-nachrs plays an important role in the cell survival . increase in intracellular calcium ions can cause indirect activation of -adrenergic receptor signaling , which induces activation of epidermal growth factor ( egf ) and activation of epidermal growth factor receptor ( egfr ) . egfr activates the akt pathway and its downstream effectors , x - linked inhibitor of apoptosis protein ( xiap)-survivin and nuclear factor-b ( nf-b ) . furthermore , nicotinic stimulation of 7-nachrs confirmed the anti - apoptotic activity of these receptors by activation of pkc , pka and nf-b , and down - regulation of the tumor suppressor p53 . nf-b phosphorylation caused by nicotine also promotes phosphorylation of the apoptotic protein bad and bax and involvement of mek and pi3k pathways . the neurotransmitter participates in numerous interrelated biologic processes such as proliferation , differentiation , apoptosis , adhesion , and migration . both acetylcholine and nicotine ( agonists of nachr ) may affect different kinds of cells by autocrine or paracrine stimulation . the cholinergic anti - inflammatory pathway is an important mechanism that inhibits cytokine production and minimizes tissue injury during inflammation . as known now , the stimulation of the vagus nerve , which releases acetylcholine , and administration of cholinergic agonists , such as nicotine and gts-21 , reduce cytokine production in the models of acute , systemic inflammation , including endotoxemia , hemorrhagic shock , ischemia- reperfusion injury , and polymicrobial sepsis . proper inhibition or stimulation of this nicotinic acetylcholine receptor ( or its selected subtype ) may give comprehensive therapeutic possibilities to effective treatment of neurodegenerative as well as inflammatory , autoimmune or cancer diseases . however , for proper application of such therapies nachr modulation in different kinds of cells should be fully understood .
acetylcholine has been well known as one of the most exemplary neurotransmitters . in humans , this versatile molecule and its synthesizing enzyme , choline acetyltransferase , 
 have been found in various non - neural tissues such as the epithelium , endothelium , mesothelium muscle , blood cells and immune cells . 
 the non - neuronal acetylcholine is accompanied by the expression of acetylcholinesterase and nicotinic / muscarinic acetylcholine receptors . 
 increasing evidence of the non - neuronal acetylcholine system found throughout the last few years has indicated this neurotransmitter as one of the major cellular signaling molecules ( associated e.g. with kinases and transcription factors activity ) . 
 this system is responsible for maintenance and optimization of the cellular function , such as proliferation , differentiation , adhesion , migration , intercellular contact and apoptosis . 
 additionally , it controls proper activity of immune cells and affects differentiation , antigen presentation or cytokine production ( both pro- and anti - inflammatory ) . 
 the present article reviews recent findings about the non - neuronal cholinergic system in the field of immune system and intracellular signaling pathways .