A novel assay to detect nucleotide receptor P2X7 genetic polymorphisms influencing numerous innate immune functions

Abstract

The importance of accessory signaling pathways amplifying endotoxin responses has recently been highlighted by genetic studies describing LPS-hyporesponsive individuals despite carrying the common allele for TLR4. The nucleotide receptor P2X7 modulates the production of numerous LPS-stimulated inflammatory mediators. We have recently described the largest phenotypic screen known for genetic polymorphisms associated with the nucleotide receptor P2X7, a global regulator of leukocyte function. This required the development of a novel monocyte pore assay with numerous advantages over previous methods and with the potential to facilitate rapid (< 3 h), multiplex analysis of clinical samples. This paper addresses aspects pertinent to the development of the monocyte pore assay, briefly summarizes our results suggesting that P2X7 alleles modulate LPS-stimulated cytokine production, and discusses a model wherein P2X7 may serve as an amplification loop of innate immunity.

Standardized method to minimize variability in a functional P2X7 flow cytometric assay for a multi‐center clinical trial

“…Samples were adjusted to 10 mM magnesium chloride, washed in HEPES-buffered saline and diluted to a volume of 2.5 mL in HBS. These latter steps have been previously shown to close the pore rapidly, allowing for kinetic precision in a large clinical study (10,30). For our previous studies in healthy subjects and the data from the Natural Cold in Asthma protocol (refs (10,30) and Table 2), the vast majority of samples were processed on the same day as the phlebotomy without inclusion of propidium iodide.…”

“…Although all leukocytes studied to date express P2X 7 , monocytes were selected as the cell population to screen because of the greater sensitivity of pore function noted between individuals participating in a small study with 45 healthy subjects (5,30). All reagents have been optimized and are prepared in large batches with aliquots sufficient for at least 200 subjects, using previously described staining procedures (10,14,30).…”

“…All reagents have been optimized and are prepared in large batches with aliquots sufficient for at least 200 subjects, using previously described staining procedures (10,14,30). Briefly, aliquots of whole blood (500 μL/aliquot) were washed twice in HEPES-buffered saline (HBS; 130 mM NaCl, 5 mM KCl, 20 mM HEPES pH 7.4, 0.1% bovine serum albumin, 10 mM glucose; components purchased at Sigma, St. Louis, MO) and resuspended in the original 500 μL volume.…”

Human P2X7 Pore Function Predicts Allele Linkage Disequilibrium

“…The design and validation of this assay for the P2X7 A1513C allele have been reported previously (30,31 ). As shown in Fig.…”

“…We previously described the design of a P2X 7 pore assay in whole blood with several features permitting high-throughput functional screening in clinical studies (30 ). 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 ).…”