The innate immune system constitutes the first line of defense against anything that compromises tissue homeostasis. Activation of innate immunity results in the induction of inflammation, which is essence, aims to restore the structural and functional integrity of tissues and organs.

Adenosine 5^′-triphosphate (ATP) is thought to be one of the DAMPs playing a major role in the inflammation and cytokine storm. It is present in high concentrations within the cytoplasm of every cell and can be released after cell damage by a variety of injurious agents 1 2 . After release, ATP acts through binding to specific receptors known as purinergic P2 receptors (P2R), of which the P2X ₇ subtype is a potent mediator of cytokine processing and release 3 .

The purinergic P2X ₇ receptor is a ligand-gated ion channel which has a wide tissue distribution, being expressed by virtually all cell types, including cells of the immune system, i.e. monocytes, macrophages, dendritic cells, and T cells 4 . Activation of this receptor by brief exposure to extracellular ATP opens a cation channel, which allows Ca²⁺ influx, as well as K⁺ efflux 5 . Longer exposure to ATP leads to dilatation of the P2X ₇ channel to a pore, which allows uptake of permeants up to the size of ethidium⁺ 6 7 . Activated P2X ₇ receptors are known to play an important role in regulating the inflammatory response in vivo (reviewed in 8 . Research indicates that activation of the P2X₇ receptor causes massive release of the pro-inflammatory mediator IL-1β. The P2X ₇ -mediated release of IL-1β by immune cells is suggested to be regulated by various mechanisms including: a) cytotoxicity of IL-1β producing immune cells 9 , b) K⁺ efflux 9 , and c) activation of the inflammasome NALP3 via pannexin-1 (reviewed in 10 ). In addition to IL-1β, other pro-inflammatory mediators are also up-regulated via P2X ₇ receptor, including IL-6, IL-18 and TNF-α (reviewed in 11 ).

These data point to an important role of P2X7 receptor-mediated signaling in inflammation, and also suggest that polymorphisms within the P2X ₇ receptor gene that lead to loss of receptor function have the potential to impair cytokine release by immune cells in vivo.

Several non-synonymous single nucleotide polymorphisms (SNP) have been characterized in the P2X ₇ receptor gene (reviewed in 12 ). One such SNP concerns the nucleotide at position 1513, which changes a glutamic acid to an alanine acid at amino position 496 (Glu496Ala). Previous research in human monocytes, showed that the Glu496Ala polymorphism decreased the P2X ₇ receptor mediated K⁺ efflux, thereby delaying P2X ₇ receptor mediated release of IL-1β 13 . Furthermore, it was shown that subjects homozygous for the variant allele of the Glu496Ala polymorphism had reduced sensitivity to inflammation compared to wild-type subjects 14 . In the present study, we further tested the hypothesis that subjects homozygous for the Glu496Ala loss-of-function polymorphism produce lower levels of IL-1β in response to ATP. In addition to levels of IL-1β, we also explored whether production of other inflammatory cytokines in response to ATP was altered in subjects carrying the Glu496Ala P2X ₇ receptor SNP.

To test our hypotheses, we used an ex vivo inflammation model by stimulating whole blood with the potent inflammatory stimuli LPS and PHA (phytohemagglutinin). Previous research showed that this whole blood assay, in contrast to isolated cells or cell lines grown in medium, closely resembles the in vivo situation and forms an appropriate and reproducible culture condition to measure cytokine production ex vivo 15 .

In vitro evidence suggests that the P2X7 receptor is involved in the induction of inflammation. In the present study, we tested the hypothesis that P2X7MUT subjects have impaired cytokine release compared to P2X7WT subjects, using an ex vivo inflammation model. In this model, blood was stimulated with the potent inflammatory stimuli LPS and PHA to trigger an inflammatory response via both the innate and adaptive arms of immunity, respectively, leading to the production of a range of different cytokines. Our group previously showed that stimulatory effects of low-level ATP (i.e. 0.3 mM) on the production of IL-10 were maximal after 24 hours of incubation, and that early inhibitory effects of 0.3 mM ATP on IL-1β, TNF-α, IL-6 and IFN-γ persisted up to this time point 16 . In the present study, whole blood from subjects with and without the Glu496Ala polymorphism in the P2X ₇ receptor gene were incubated with ATP concentrations ranging from 0.3 to 3 mM to evaluate effects of this loss-of-function polymorphism on cytokine release via P2X ₇ .

Contrary to our hypothesis, LPS/PHA-induced IL-1β release showed a concentration-dependent stimulation by ATP incubation of blood from P2X7MUT subjects, whereas ATP induced a concentration-dependent attenuation of the LPS/PHA-induced IL-1β release in P2X7WT subjects. However, the most striking result observed in the present study was that release of IL-1β as well as release of TNF-α, IL-6, IL-10 and IFN-γ was almost completely abolished in whole blood from P2X7WT subjects after 24-hour LPS/PHA stimulation in the presence of 3 mM ATP, whereas such an effect was not observed in P2X7MUT subjects.

It is known that apoptosis, which is a morphologically distinct form of cell death 17 , is induced in immune cells by prolonged or excessive activation of the P2X ₇ receptor by high levels of ATP 18 . Since impaired P2X ₇ receptor function in leucocytes from subjects with the Glu496Ala polymorphism could lead to resistance to P2X ₇ -mediated apoptosis, we measured LDH levels as an indicator of ATP-induced cell death in the present study. Results showed increased LDH levels after 24-hour exposure to high concentrations of ATP in P2X7WT subjects, but not in P2X7MUT subjects. This finding, combined with the striking observation that levels of all cytokines were almost completely abolished inP2X7WT subjects after 24-hour LPS/PHA stimulation of whole blood in the presence of 3 mM ATP, would suggest that leucocytes from P2X7MUT subjects were protected against ATP-induced cell death, presumably via impaired P2X ₇ -mediated apoptosis. This result is in line with a previous study, which showed a marked reduction in non-viable lymphocytes in P2X7MUT subjects 7 , strengthening the hypothesis that the Glu496Ala polymorphism indeed causes resistance to apoptosis.

In studies in isolated cell lines, it has been well established that extra cellular ATP at high concentrations triggers massive release of IL-1β, thereby inducing a strong pro-inflammatory response 19 20 . Numerous in vitro studies as well as in vivo studies with P2X ₇ receptor knock-out mice have demonstrated that the P2X ₇ receptor is the main receptor responsible for the release of IL-1β induced by ATP 21 22 23 . Therefore, polymorphisms is the P2X7 receptor gene causing a function change in the P2X ₇ receptor, such as the Glu496Ala polymorphism, could lead to impaired IL-1β releases. Sluyter et al. 13 previously showed that in whole blood from subjects harbouring the Glu496Ala polymorphism IL-1β release was 78% lower than in whole blood from wild-type subjects after 30 min of 6 mM ATP treatment. However, after 60 min ATP treatment this inhibition of IL-1β release was no longer apparent 13 . More recently, also a 6-hour incubation with 1mM ATP did not induce an attenuation of IL-1β release in P2X7MUT subjects 14 . It has been demonstrated that the Glu496Ala polymorphism results in impaired ATP-induced pore formation, but does not cause a total loss of P2X ₇ channel function 7 . Indeed, Sluyter et al 24 found that P2X7MUT subjects showed a reduced but not totally abolished P2X ₇ receptor-mediated K⁺ efflux compared to P2X7WT subjects. The authors suggested that the smaller residual K⁺ efflux may still be sufficient to cause delayed release of IL-1β in subjects harbouring the Glu496Ala polymorphism, offering a putative explanation why no reduction in IL-1β release has been observed after prolonged incubation with ATP in monocytes from these subjects. In our study, we observed even increased levels of IL-1β in P2X7MUT subjects compared to P2X7WT subjects after 24-hour ATP incubation. This result might be explained by the fact that the Glu496Ala SNP incompletely impairs P2X ₇ receptor function, that is, delaying but not abolishing the K⁺ efflux that is essential for IL-1β release, while impairing pore formation is essential for apoptosis. Another possible explanation for the observed increased IL-1β release in P2X7MUT subjects might be the activation of immune cells by other proinflammatory cytokines, such as TNF-α and IFN-γ 25 26 . It is difficult, however, to draw conclusions about differences between P2X7WT vs. P2X7MUT subjects regarding effects of ATP at 3 mM on production of IL-1β as well as production of other cytokines, since the effects of ATP at these concentrations in our study appeared to be distorted by cytotoxic effects via P2X ₇ in P2X ₇ WT subjects.

Previous findings showed the involvement of the P2X ₇ receptor in the ATP-induced secretion of IL-6 in several diseases 27 . In the present study, however, decreased levels of IL-6 were observed after incubation of LPS/PHA stimulated blood with ATP. Furthermore, this decrease was less pronounced in P2X7MUT subjects compared to P2X7WT subjects. Future research on the effects of ATP induced release of IL-6, and the involvement of the P2X ₇ receptor in IL-6 release is therefore warranted.

In a previous study by our group in which we used the whole blood assay to test the influence of ATP on the release of a number of cytokines 16 , it was shown that co-incubation of whole blood from healthy volunteers with LPS/PHA and 0.3 mM ATP attenuated the rise of the pro-inflammatory cytokine TNF-α and stimulated the secretion of the anti-inflammatory cytokine IL-10. We demonstrated pharmacologically 28 that the stimulation of TNF-α release and the inhibition of IL-10 release were due to ATP-induced activation of the P2Y₁₁and P2Y₁₂ receptor subtypes, respectively. In agreement with these earlier studies, we found in the present study that ATP at 0.3 mM decreased TNF-α levels in both P2X7WT and P2X7MUT subjects. Although pharmacological evidence in rat microglia suggests that TNF-α levels may be increased via activation of the P2X ₇ receptor 29 , our data would rather suggest that the P2X ₇ receptor is not involved in the regulation of TNF-α release in whole blood ex vivo at low levels of ATP.

Little is known about the direct regulation of production of IL-10 by the P2X ₇ receptor. Denlinger et al. 14 showed that P2X7MUT subjects released higher levels of IL-10 in response to ATP than P2X7WT subjects. Our data, however, do not show a differential effect of ATP at 0.3 and 0.9mM on IL-10 in P2X7MUT vs. P2X7WT subjects.

It is known that hemolysis can significantly increase plasma ATP concentration 30 . Therefore, the differences between P2X7MUT and P2X7 WT subjects found in the present study could have been confounded by the different amount of hemolysis in the samples of these subjects. However, our HPLC data shows that the plasma ATP levels of P2X7MUT and P2X7 WT were similar and are unlikely to have influenced our results.

In this study we only focused on the role of the P2X7 receptor subtype in the inflammatory response. Several other members of the P2 purinergic receptor family have been shown to play a in the ATP induced inflammatory response 31 . Moreover, recent evidence has indicated a role for the P2X4 receptor subtype in the regulation of P2X7-mediated inflammatory functions 32 . Therefore, our results might have been influenced by the presence of non-synonymous SNPs within other P2X receptor genes causing functional changes of these receptors. In the body, an interplay exists between bone homeostasis and immune processes. Research dealing with crosstalk between the bone and immune system, i.e. the field of osteoimmunology, is receiving growing attention 33 34 . Bone marrow is the principal site of haematopoiesis allowing a close interaction between bone and immune cells. Cytokines released during immune responses are known to affect the bone metabolism, and dysregulated cytokine responses are implicated in bone disease such as osteoporosis. IL-1β as well as TNF-α and IL-6 are known to promote osteoclastogenesis either by increasing osteoclast generation and activation or by inducing RANKL expression by osteoblasts 35 36 , whereas IL-10 and IFN-γ are known to be inhibitors of osteoclastogenesis by blocking RANKL signalling, either directly or indirectly 35 . Alterations in cytokine release due to the Glu496Ala polymorphisms leading to loss of P2X ₇ receptor function might therefore contribute to the development of osteoporosis. As we observed increased levels of both IL-1β and TNF-α in P2X7MUT subjects relative to P2X7WT subjects, it might be suggested that P2X7MUT subjects have an increased risk to develop osteoporosis. This hypothesis is consistent with previous reports showing that the Glu496Ala polymorphism, leading to a non-functional P2X ₇ receptor, is associated with decreased BMD values, i.e. increased osteoporosis risk 37 38 39 .

In conclusion, a difference in the ATP-induced cytokine release and cell-death was observed between P2X7MUT subjects and P2X7WT subjects, indicating that the Glu496Ala loss-of-function polymorphism is likely to play an important during inflammation. Therefore, the P2X7 receptor might be of importance in the aetiology and pathophysiology of inflammatory diseases as well as related conditions, such as osteoporosis. However, further research to unravel the exact role of non-synonymous SNPs within the P2X ₇ receptor gene is warranted.

The authors declare that they have no competing interests.

AW and ET carried out the experiment. AW and MB analyzed data and performed the statistical analysis. I. Arts, PD and PG conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.