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P2X receptors (P2XRs) are membrane ligand-gated ion channels and are members of the purinergic receptor family. Of the seven P2XR family members, only four of them (P2X1, P2X4, P2X6 and P2X7) have been shown to be expressed in MCs, with each of them playing an important role in regulating MC activities, such as Ca+ influx and degranulation. P2XRs are also present in neurons and glial cells, where their engagement may affect the development of neuroinflammatory pathologies such as the Alzheimer’s disease (AD), Parkinson’s disease (PD) and Multiple sclerosis (MS).
- mast cells
- microglia
- astrocytes
- oligodendrocytes
- P2X receptors
1. Introduction
Mast cells (MCs) are immune cells that form part of the innate branch of the immune system. Since MCs express a broad spectrum of high affinity receptors (e.g., high-affinity IgE receptor (FcɛRI), Fc-gamma receptor (FcγR), complement receptors and purinergic receptors), they rapidly respond to a variety of environmental and immune stimuli, resulting in the release of pre-formed mediators such as histamine and then later the production of newly synthesized cytokines, chemokines, growth factors, proteases, and lipid mediators [1,2,3].
MCs are of dual hematopoietic origin. In mice, a first wave of MCs originates during embryonic development from yolk-sac progenitors followed by a second wave of bone marrow-derived MCs in adulthood. This duality in the origin of MCs may influence the cell phenotype and function in various tissues [4,5]. After leaving the bone marrow, MC committed progenitors circulate in the bloodstream and mature in peripheral tissues under the influence of a cocktail of growth factors that include the stem cell factor [6,7].
Human MCs are found in low numbers in the hypothalamus, leptomeninges, area postrema, and the dura matter of the spinal cord [8]. Nearly 97% of all MCs found in the brain are positioned in the abluminal side of the brain blood vessels, which allows them to communicate with neurons, glial cells (such as astrocytes and microglia) and endothelial cells [8,9]. In the human brain, MC density was found to be less than <5 MCs in 5 μm thick tissue sections in meninges and perivascular area. However, during viral, bacterial and parasitic infections, MC numbers were observed to be higher, at around 11–20 cells per 5 μm thick tissue section in meninges and around 5–20 in perivascular area [10]. In the healthy brain the predominant MC phenotype is tryptase+ chymase+ and their overall numbers can be affected by trauma and/or stress, whilst their activation could potentially influence social behaviour [9,10,11,12]. The number of MCs in the brain can be sex-dependent, especially in young mice pups, where the total number of MCs in the preoptic area is nearly two times higher for males than females, potentially contributing to the gender bias in human neuropathology for diseases such as autism spectrum disorder (ASD) or schizophrenia [13,14]. MC numbers are also age-dependent, with MCs being most abundant in brains of individuals under 19 years old, a pattern probably related to the age involution [8,15,16].
2. Expression of P2XRs and the Role of ATP and P2XR Activation in Neuroinflammation
2.1. ATP Release during Inflammation and Brain Pathology
In the CNS, extracellular ATP acts as a fast-excitatory neurotransmitter and an important mediator for neuron-glial, glial-glial, and neuron-neuron communication [62,63]. In a healthy tissue, ATP is found extracellularly at negligible concentrations, with neurons and glial cells carrying millimolar concentrations of ATP intracellularly [64]. This is released by Panx1 and Connexin channels, through vesicular transport or through membrane stress/damage [65]. For example, during inflammation, necrotic cells set free up to hundreds of µmol/L of ATP [66]. Increased concentrations of ATP have been detected in brain pathologies upon trauma, ischemia, epilepsy, PD or MS [67,68,69].
2.2. Expression of P2XRs and The Role of ATP and P2XR Activation in Glial Cells
Extracellular ATP is the sole activator of all P2XR family members and undergoes a rapid enzymatic degradation into adenosine diphosphate (ADP) upon extracellular release, which is then further degraded into adenosine monophosphate (AMP) and adenosine [70,71]. P2XR engagement by ATP activates Na+ and Ca2+ influx, and K+ efflux, resulting in the plasma membrane depolarisation and reorganization, release of cytokines (such as IL-6, IL-8 and TNF-α) and caspase activation [72,73]. The seven P2XRs exhibit different affinities for ATP and exist as homomeric or heteromeric receptors, with homomeric P2X5 and P2X6 potentially not being functional in humans. For P2X6 in particular heteromerization appears to be necessary for a correct folding and assembly [74,75,76].
Together with the P2XRs, eight different P2YRs belong to the purinergic receptor family. P2YRs are G-protein coupling receptors expressed and functional on MCs and glial cells and involved in AD or epilepsy disease pathogenesis [77,78,79,80]. However, since the P2YRs are activated by multiple mediators, such as ATP, ADP, UTP, UDP and UDP-glucose [81] and since this review only examines the unique role of ATP in linking glial and MC activities, these receptors will not be discussed further.
2.3. P2XR Expression in Astrocytes
In astrocytes, P2X1 exists as a homomeric, or as a P2X1/P2X5 heteromeric receptor, the latter having unique properties compared to its homomeric counterpart. For example, the P2X1/P2X5 heteromeric receptor in astrocytes has a higher sensitivity to ATP with no desensitization response compared to the homomeric P2X1 [82,83]. However, P2X1/P2X5 expression is age-dependent, with a lack of heterodimers in 6 month old mice [22]. It is therefore unlikely that this receptor has an impact on astrocytes activities in adult mice.
P2X2 activation in astrocytes was found to regulate GABAergic transmission and ASD like behaviour in C57BL/6J mice carrying a knockout of the type 2 inositol 1,4,5-trisphosphate 6 receptors (IP3R2) gene, as mutations in this gene are associated with ASD [84]. Activation of the P2X2 also led to an increase in mRNA expression of leukaemia inhibitory factor (LIF), a cytokine inhibiting cell differentiation, in astrocytes isolated from neonatal C57BL/6J mice. Thus, this contributing to the efficacy of electroconvulsive therapy in psychiatric disorders [85].
Expression of P2X3 was reported in astrocytes in Sprague-Dawley rats [86] and in primary astrocytes cultures obtained from rats cerebral cortex [87], with receptor activation modulating craniofacial neuropathic pain [88].
Regarding P2X4, there is still limited evidence of its expression in astrocytes. This was demonstrated by RT-PCR and immunohistochemistry in rat cells [89,90]. However, studies performed in GFAP promoter-controlled EGFP-expressing (GFAP/EGFP) transgenic mice and in vitro using hippocampal slices from transgenic GFAP/EGFP mice and Wistar rats did not detect any P2X4-mediated ATP-induced current [82,91].
Rat cortical astrocytes [87] and human astrocytes isolated from foetal cortex express P2X5 [92]. However, knowledge about the receptor functionality is restricted to the P2X1/P2X5 heterodimers [80].
The presence of P2X6 in astrocytes remains controversial, as RT-PCR and western blotting of primary astrocytes from rats cerebral cortex didn’t show any expression [87], whilst P2X6 expression was detected using qPCR in human astrocytes from foetal cortex [92] and in astrocytes end-feet derived from Sprague-Dawley rats [93].
P2X7 activation was shown to attenuate LPS-induced release of TNF-α in primary cultures of rat cortical astrocytes [94]. In contrast, stimulation of P2X7 in mouse astrocyte cultures resulted in the secretion of various transmitter molecules, such as glutamate or GABA [95], and of a MAP kinase-controlled secretion of CCL2 in the Sprague-Dawley rat astrocytes [96]. In the hippocampus of C57BL/6J mice, P2X7 activation with extracellular ATP resulted in the release of neurotransmitters from astrocytes, leading to the stimulation of surrounding neurons [97]. In human foetal astrocyte cultures, regulation of P2X7 was induced by IL-1β [98] and its expression was observed in astrocytes from post-mortem brain tissues sections in AD patients [99]. In SOD1 mice astrocytes, P2X7 activation contributed to their toxicity towards motor neurons [100].
2.4. P2XR Expression in Microglia
In both mouse and human microglia, P2X4 and P2X7 are highly expressed [101], while evidence for the expression of P2X1, P2X2, P2X3 and P2X6 remains controversial. In this regard, microglia cultures obtained from Sprague-Dawley rats and BV-2 (immortalized murine microglia) cells showed P2X1 expression [102,103], whilst C57BL/6J and SOD1 mice microglia cells displayed very low or no expression [104,105]. Xiang & Burnstock [106] showed P2X1 expression in Wistar rats microglia only at late stages of embryonic development and until day 30 of postnatal development, suggesting that the expression of P2X1 in animal models might be species and age dependent. In human microglia, voltage-clamp electrophysiology performed after ATP stimulation in two donors showed no evidence of rapid desensitising inward current expected from P2X1 and P2X3 engagement [107]. RNA sequencing studies by Chiu et al. [108] and Solga et al. [109] in microglia from SOD1 and C57BL/6 mice, detected either none or extremely low expression levels of P2X1, P2X2, P2X3, P2X5 and P2X6, respectively. On the contrary, western blot analysis of N9 murine microglial cell line showed the presence of P2X1, P2X2, P2X3 and P2X6 [110]. Thus, discrepancies in P2X1, P2X2, P2X3 and P2X6 expression were found not only between species but also in similar murine systems.
P2X4 plays a major role in the regulation of neuronal and glial functions, as peripheral damage induces an upregulation of P2X4 in microglia and affects the inflammatory response [111]. It appears that P2X4 in rat cultured microglia is predominantly stored intracellularly while membrane expression is rapidly upregulated through C-C chemokine receptor type (CCR) 2-mediated activation upon CCL2 or CCL12 ligand binding [112]. Stimulation of P2X4 in mice microglia leads to maintained mechanical hypersensitivity after nerve injury, through the release of brain-derived neurotrophic factor [113], which is a crucial signalling mediator between microglia and neurons [114]. Deletion of P2X4 in P2X4−/− KO mice resulted in the absence of mechanical hypersensitivity after peripheral nerve lesion [113]. In a mouse model of experimental autoimmune encephalomyelitis (EAE), P2X4 was shown to be a modulator of microglia polarization and its increase in expression to be a marker of the neuroinflammatory response [115]. Furthermore, P2X4 was also suggested to contribute to the activation and migration of Lewis rat microglia into the site of a formalin-induced injury [116].
Activation of the P2X7 in mice microglia results in the activation of the inflammasome, release of TNF-α, CCL2, IL-6, IL-1β, and IL-18, and increased cell death [117,118]. In healthy human donors, microglia isolated from the cortex expressed functional P2X7, but no release of IL-1β or IL-18 was observed upon LPS priming and subsequent ATP stimulation. The authors hypothesized that the cultured cells switched from a M1 inflammatory phenotype to an anti-inflammatory M2 phenotype in the presence of serum contained in the culture medium, therefore possibly shifting the nature of the microglia behaviour [107].
P2X7 activity in microglia has been linked to several neuroinflammatory diseases. In a mouse model of AD, upregulation of P2X7 expression was observed in microglia in proximity to Aβ peptide aggregates, and this expression was further elevated in the later stages of Aβ pathology. The same results were then observed in AD patients, suggesting an importance of P2X7 in AD pathology [23,99,119]. P2X7 activation in microglia has also been linked to MS, stress, depression, and PD in Sprague-Dawley rats, Wistar rats and C57BL/6J mice models [24,25,120,121]. Upregulation of P2X7 expression in microglia was observed in SOD1 mice [122] and receptor activation was found to modulate autophagic flux, a homeostatic mechanism involved in degradation of damaged organelles and protein aggregates, whose abnormalities were reported in ASL [123]. Inhibition of P2X7 using brilliant blue G showed prolonged survival in female SOD1 mice [124], while administration of JNJ-47965567 P2X7 inhibitor in the same model did not alter ALS progression [125].
2.5. P2XR Expression in Oligodendrocytes
Expression of P2X1, P2X2 and P2X3 was observed in oligodendrocytes progenitor cells isolated from postnatal 1 day Wistar rats [126] and in human stem cell-derived oligodendrocytes progenitor cells [127], while was absent in mouse mature and progenitor oligodendrocytes [128].
P2X4 expression in oligodendrocytes was confirmed by western blot analysis, qPCR and RNA sequencing in mice, rats and human progenitor oligodendrocytes [126,127,128]. However, Zabala et al. [115] were unable to link receptor expression to functionality in these cells. P2X5 and P2X6 were not found in oligodendrocytes lineage cells [129].
In contrast to other P2XRs, the functionality of P2X7 in oligodendrocytes has been demonstrated by Matute et al. [130] where a continuous activation of P2X7 led to the oligodendrocytes’ death, due to the P2X7-mediated Ca2+ toxicity. Furthermore, an increase in P2X7 expression in oligodendrocytes was found in samples from patients with MS, in a mouse model in post-episodes of status epilepticus and during epilepsy [131], and in rat model of ischemic damage [132].
2.6. Expression of P2XRs and The Role of ATP and P2XR Activation in MCs
In MCs, expression of P2X1, P2X4 and P2X7 has been confirmed by RT-PCR analysis in LAD2 cells and human lung MCs, and by proteomics analysis in human and mouse primary connective tissue MCs [19,134]. P2X6 expression has also been observed in LAD2 and human lung MCs, however its functionality has not been demonstrated yet [19,135].
The MC homomeric P2X1 binds ATP with high affinity, with only 1 μM needed to activate P2X1 in LAD2 cells [19]. Study by Wareham & Seward [20] observed that the engagement of the P2X1 triggers a fast and transient calcium influx and a prolonged exposure to even low ATP concentrations may lead to its desensitisation. Even though it was concluded that P2X1 activation in LAD2 cells does not trigger degranulation, it was not investigated further if the activation might lead to a release of specific mediators.
Like the P2X1, P2X4 activation leads to a calcium influx into MCs without inducing degranulation. However, P2X4 activation with an ATP concentration of less than 300 μM significantly increased degranulation mediated by high-affinity IgE receptor or by G-coupled prostaglandin EP3 receptor stimulation in bone marrow-derived MCs (BMMCs) from C57BL/6 mice [21,136]. P2X4 stimulation by ATP was also shown to enhance antigen-induced phosphorylation of Syk and PLCγ signalling pathways in mice BMMCs, independent of the P2X4-mediated calcium influx [137]. Inhibition of P2X4, by the potent and selective benzodiazepine derivative 5-BDBD, in human lung MCs diminished release of cysteinyl leukotrienes [135].
To date, P2X7 is the only P2XR demonstrated to induce MC degranulation. Activation of P2X7 triggers degranulation of meningeal MCs derived from C57BL/6 mice and human LAD2 cells [20,138], resulting in the immediate release of many pre-stored inflammatory mediators (such as histamine, tryptase, chymase, IL-6, IL-1β, and CCL2), with others, such as IL-5, CCL3 and eicosanoids, being newly synthesised over time [139,140]. Furthermore Shimokawa et al. [141] reported that P2X7 activation by ATP induced the secretion of IL-33 in mouse BMMCs.
2.7. Expression of P2XRs: Public Gene Expression Databases
The availability of public datasets such as the iFANTOM and ImmGen consortiums now provide a useful tool to glean additional information on the expression of P2XRs in MCs and glial cells. P2X1, P2X4 and P2X7 are mostly expressed in the peritoneal cavity while P2X2, P2X3, P2X5 and P2X6 expression is constitutive and shared between organs and tissues. It should be noted, that P2XR expression is lower in MCs and glial cells compared to other cell types, except for P2X1, which exhibits the highest expression in skin MCs.
This entry is adapted from the peer-reviewed paper 10.3390/cells10092282
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