Human neutrophils do not express purinergic P2X7 receptors
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It has been reported that in human neutrophils, external ATP activates plasma membrane purinergic P2X7 receptors (P2X7R) to elicit Ca2+ entry, production of reactive oxygen species (ROS), processing and release of pro-inflammatory cytokines, shedding of adhesion molecules and uptake of large molecules. However, the expression of P2X7R at the plasma membrane of neutrophils has also been questioned since these putative responses are not always reproduced. In this work, we used electrophysiological recordings to measure functional responses associated with the activation of membrane receptors, spectrofluorometric measurements of ROS production and ethidium bromide uptake to asses coupling of P2X7R activation to downstream effectors, immune-labelling of P2X7R using a fluorescein isothiocyanate-conjugated antibody to detect the receptors at the plasma membrane, RT-PCR to determine mRNA expression of P2X7R and Western blot to determine protein expression in neutrophils and HL-60 cells. None of these assays reported the presence of P2X7R in the plasma membrane of neutrophils and non-differentiated or differentiated HL-60 cells—a model cell for human neutrophils. We concluded that P2X7R are not present at plasma membrane of human neutrophils and that the putative physiological responses triggered by external ATP should be reconsidered.
KeywordsHuman Neutrophils Purinergic receptors Protein Whole cell current
We wish to thank Carmen Y. Hernandez-Carballo for excellent technical assistance. We are grateful to Drs. Elias Pérez and Rafael Rubio for allowing us use their spectrofluorometer and the fluorescence microscope. This work was supported by grants from CONACyT, Mexico (59889 to JA, 79897 to JA, 45895 to PPC) and NIH, USA (PO1-HL18208 to R. Waugh and JA). Drs. Teresa Rosales-Saavedra and Carmen Toro-Castillo held a Postdoctoral Fellowship from CONACyT (Mexico); Griselda Casas-Pruneda and Guadalupe Martel-Gallegos hold a PhD fellowship from CONACyT (Mexico).
- 3.Axtell RA, Sandborg RR, Smolen JE, Ward PA, Boxer LA (1990) Exposure of human neutrophils to exogenous nucleotides causes elevation in intracellular calcium, transmembrane calcium fluxes, and an alteration of a cytosolic factor resulting in enhanced superoxide production in response to FMLP and arachidonic acid. Blood 75:1324–1332PubMedGoogle Scholar
- 4.Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M (1983) Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol 4:1910–1917Google Scholar
- 18.Gasmi L, McLennan AG, Edwards SW (1996) The diadenosine polyphosphates Ap3A and Ap4A and adenosine triphosphate interact with granulocyte-macrophage colony-stimulating factor to delay neutrophil apoptosis: implications for neutrophil: platelet interactions during inflammation. Blood 87:3442–3449PubMedGoogle Scholar
- 19.Gu BJ, Zhang WY, Bendall LJ, Chessell IP, Buell GN, Wiley JS (2000) Expression of P2X7 purinoceptors on human lymphocytes and monocytes: evidence for nonfunctional P2X7 receptors. Am J Physiol 279:C1189–197Google Scholar
- 23.Kaba NK, Schultz J, Law FY, Lefort CT, Martel-Gallegos G, Kim M, Waugh RE, Arreola J, Knauf PA (2008) Inhibition of Na+/H+ exchanger enhances low pH-induced L-selectin shedding and beta2-integrin surface expression in human neutrophils. Am J Physiol Cell Physiol 295:C1454–C1463CrossRefPubMedGoogle Scholar
- 30.Miyabe K, Sakamoto N, Wu YH, Mori N, Sakamoto H (2004) Effects of platelet release products on neutrophilic phagocytosis and complement receptors. Thromb Res 114:23–36Google Scholar