Detection of human P2X7 nucleotide receptor polymorphisms by a novel monocyte pore assay predictive of alterations in lipopolysaccharide-induced cytokine production.
The nucleotide receptor P2X(7) 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 P2X(7) gene predispose to the production of an anti-inflammatory mediator balance. Accordingly, we developed a novel P2X(7) 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 P2X(7) 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-alpha (p = 0.036) and higher amounts of IL-10 (p < 0.001) relative to the high pore controls. This reduction in the TNF-alpha to IL-10 ratio persisted to at least 24 h and is further decreased by cotreatment with 2-methylthio-ATP. The ability of P2X(7) polymorphisms to regulate the LPS-induced TNF-alpha 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 P2X(7) pore activity.
Denlinger
Loren C
LC
Department of Medicine, University of Wisconsin Medical School, Madison, WI 53792, USA. ldenling@wisc.edu <ldenling@wisc.edu>
Angelini
Giuditta
G
Schell
Kathleen
K
Green
Dawn N
DN
Guadarrama
Arturo G
AG
Prabhu
Usha
U
Coursin
Douglas B
DB
Bertics
Paul J
PJ
Hogan
Kirk
K
D000293:Adolescent; D000328:Adult; D002463:Cell Membrane Permeability; D016207:Cytokines / Q000096:biosynthesis*; D004252:DNA Mutational Analysis; D006801:Humans; D007111:Immunity, Cellular / Q000235:genetics*; D007239:Infections / Q000276:immunology; D007249:Inflammation / Q000235:genetics; D016753:Interleukin-10 / Q000097:blood; D008070:Lipopolysaccharides / Q000494:pharmacology; D008403:Mass Screening; D008875:Middle Aged; D009000:Monocytes / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D011110:Polymorphism, Genetic / Q000502:physiology*; D011237:Predictive Value of Tests; D018048:Receptors, Purinergic P2 / Q000235:genetics* / Q000502:physiology; D058486:Receptors, Purinergic P2X7; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis
D016207:Cytokines; D008070:Lipopolysaccharides; C546468:P2RX7 protein, human; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10