Short communicationActivation of the damage-associated molecular pattern receptor P2X7 induces interleukin-1β release from canine monocytes
Introduction
Damage-associated molecular patterns (DAMPs) play important roles in inflammation and immunity by functioning as signals of cell damage, stress or death during infection, injury or disease (Kono and Rock, 2008, Chen and Nuñez, 2010). One of the best-characterised DAMP is extracellular adenosine 5′-triphosphate (ATP), which mediates its effects through the activation of the P2X7 purinergic receptor, a trimeric ATP-gated cation channel (Bours et al., 2011, Wiley et al., 2011). Activation of P2X7 by extracellular ATP causes the flux of Ca2+, Na+ and K+, as well as the uptake of organic cations such as ethidium+ and YO-PRO-12+ (Jarvis and Khakh, 2009). Furthermore, P2X7 activation induces various downstream events including the NALP3 inflammasome-dependent maturation of interleukin (IL)-1β, and its subsequent release from various myeloid cell types (Di Virgilio, 2007). This event, at least in monocytes, requires the prior activation of cells with the TLR4 ligand, LPS, which results in the up-regulation and assembly of the NALP3 inflammasome, as well as the synthesis of IL-1β (Mehta et al., 2001, Bauernfeind et al., 2009). Due to this and other properties of P2X7 activation, this receptor plays important roles in human health and disease (Sluyter and Stokes, 2011), and thus may have similar roles in canine health and disease.
The presence of functional P2X7 on human and murine cell types is well established, but little is known about P2X7 in other mammalian species including the dog. We have previously demonstrated that peripheral blood monocytes, lymphocytes and erythrocytes from English Springer Spaniels express functional P2X7 (Sluyter et al., 2007, Shemon et al., 2008, Stevenson et al., 2009). In particular, we have previously shown that the relative amount of P2X7 function in monocytes is about half that of canine T cells but similar to that of B cells (Stevenson et al., 2009). Similar studies in other dog breeds however are lacking, and given that distinct phenotypic traits exist between breeds it remains necessary to examine P2X7 in additional breeds. Moreover, it remains unknown if P2X7 activation can induce the release of IL-1β from canine monocytes, and there is a general paucity of studies examining IL-1β release from canine myeloid cell types. Given the importance of P2X7 in human health and disease (Sluyter and Stokes, 2011), new knowledge about this receptor and events downstream of its activation in the dog is necessary to establish and understand the role of P2X7 in canine health and disease. Therefore, we investigated P2X7 in canine monocytes, and in particular its role in the release of IL-1β.
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Materials
Ficoll-Paque™ PLUS was from GE Healthcare Biosciences (Uppsala, Sweden). A438079 was from Tocris Bioscience (Ellisville, MO). ATP and LPS (Escherichia coli serotype 055:B5) were from Sigma Chemical Co (St. Louis, MO). YO-PRO®-1 iodide solution, RPMI-1640 medium, L-glutamine and ExoSAP-IT were from Invitrogen (Grand Island, NJ). FCS was from Bovogen Biologicals (East Keilor, Australia). BigDye Terminator v3.1 was from Applied Biosystems (Carlsbad, CA).
Canine monocytes
Peripheral blood was collected into VACUETTE®
P2X7 activation induces organic cation uptake into peripheral blood monocytes
To determine if P2X7 activation induces the uptake of an organic cation into monocytes from additional dog breeds to English Springer Spaniels, ATP-induced YO-PRO-12+ (375 Da) uptake into peripheral blood monocytes from two Staffordshire Bull Terriers and one Bull Terrier was examined in the absence or presence of A438079, which impairs human, monkey, rat and murine P2X7 (Donnelly-Roberts et al., 2009, Bradley et al., 2011). In the absence of A438079, ATP induced YO-PRO-12+ uptake into
Conflict of interest
The authors have no conflict of interest to declare.
Acknowledgements
We gratefully acknowledged helpful advice from Jason McArthur and Mark Dowton (both University of Wollongong), and excellent technical assistance by Margaret Phillips (University of Wollongong) and the staff of the Illawarra Health and Medical Research Institute. This work was kindly supported by grants from the American Kennel Club Canine Health Foundation and the Canine Research Foundation. The sponsors did not have any influence in study design; in the collection, analysis and interpretation
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Current address: Department of Pharmacology, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, NSW 2052, Australia.
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These authors contributed equally to this work.