Detection of Human P2X7 Nucleotide Receptor Polymorphisms by a Novel Monocyte Pore Assay Predictive of Alterations in Lipopolysaccharide-Induced Cytokine Production

Abstract

The nucleotide receptor P2X7 is expressed by most leukocytes and initiates signaling events that amplify numerous LPS responses. We tested the hypothesis that loss-of-function polymorphisms in the human P2X7 gene predispose to the production of an anti-inflammatory mediator balance. Accordingly, we developed a novel P2X7 pore assay in whole blood that magnifies the activity from wild-type alleles and preserves the gene dosage effect for the 1513 C polymorphism (AA, 69 ± 4; AC, 42 ± 4; and CC, 6 ± 1-fold stimulation). Thirty of 200 healthy individuals were identified as having low P2X7 pore activity. Seven low pore subjects were 1513 CC, 3 and 11 participants had the other known variants 946 GA and 1729 TA respectively; the remaining 9 volunteers likely have novel polymorphisms. Because platelets are a large source of extracellular ATP during inflammation, whole blood was treated ex vivo with Salmonella typhimurium LPS in the absence of exogenous nucleotides. LPS-stimulated whole blood from individuals in the low pore activity group generated reduced plasma levels of TNF-α (p = 0.036) and higher amounts of IL-10 (p < 0.001) relative to the high pore controls. This reduction in the TNF-α to IL-10 ratio persisted to at least 24 h and is further decreased by cotreatment with 2-methylthio-ATP. The ability of P2X7 polymorphisms to regulate the LPS-induced TNF-α to IL-10 ratio suggests that 15% of healthy adults may exhibit anti-inflammatory mediator responses during major infectious perturbations of the immune system, which can be predicted by P2X7 pore activity.

Human P2X7 Pore Function Predicts Allele Linkage Disequilibrium

“…Recruitment of 200 healthy persons, P2X7 A1513C allele frequencies, and the effects of low pore activity on LPS-induced cytokine generation in whole blood have been reported previously (31 ).…”

“…With this method, the optimal threshold is selected as the point of the ROC curve demonstrating a 45-degree tangent slope (32 ). P2X7 genotyping and allele detection DNA was harvested from whole blood as described previously (31 ). In addition to the evaluation of the pore assay performance characteristics mentioned above, the following methods for data acquisition and analysis are specific to this report.…”

“…The whole-blood P2X 7 pore assay evaluating 2Ј-3Ј-O-(4-benzoylbenzoyl (Bz)-ATP-stimulated YO-PRO-1 uptake by CD14ϩ monocytes has been described previously (31 ). Genotypes for the validated loss-of-function P2X7 alleles G946A, A1513C, and T1729A were determined for all 200 participants in the screening phase, as described below.…”

“…This assay preserves the allele dose dependency expected for the A1513C variant and correlates with the ratio of LPS-stimulated TNF-␣ to IL-10 in whole blood after 6 and 24 h of treatment (31 ). In the present study, we hypothesized that evaluation of persons with discordance between P2X7 genotype and pore status would disclose additional alleles, linkage disequilibrium, and novel functional correlates of genotype to phenotype.…”

Cell signaling via the P2X7 nucleotide receptor: linkage to ROS production, gene transcription, and receptor trafficking

“…In this regard, several single nucleotide polymorphisms (SNPs) have been identified in the promoter, introns, and coding sequences of P2X 7 [29,[32][33][34][35][36][37][38][39][40][41]. Investigations evaluating the significance of P2X 7 polymorphisms and overall protein expression levels support the idea that cells need to correctly respond to extracellular ATP via P2X 7 for the mediation of several functions, including neurotransmission, mycobacterial killing, and appropriate cell death.…”