Development of a native whole blood assay to measure antagonist effects on ATP-stimulated interleukin-1β release from human and dog blood

Shilina Roman, Elena Fonfria, Iain.P. Chessell, Anton.D. Michel
GlaxoSmithkline, Harlow, Essex, United Kingdom

The ATP-gated P2X7 receptor is of considerable therapeutic interest due to its pivitol role in stimulating the processing and release of interleukin-1β (IL-1β) from activated inflammatory cells. ATP stimulated IL-1β release from lipopolysaccharide (LPS)-primed human whole blood has been demonstrated previously and suggested to be mediated via the P2X7 receptor (Labasi et al., 2002). In this study we have adapted this method for assessing the potency of P2X7 antagonists in human and dog whole blood.

Whole blood was collected from human volunteers and dogs into citrate buffer (15%) following standard ethical procedures. Blood (80μl) was stimulated (1μg.ml-1 LPS) for 120 min at 37°C and then ATP (20μl) added and incubations continued for a further 10-60 min. Reactions were stopped with ice cold RPMI medium. In antagonist studies, whole blood was pre-incubated with antagonist for 40 min prior to ATP addition. Plasma supernatants were harvested and their content of mature IL-1β was determined using a bioassay based on the ability of IL-1β to activate an endogenous human IL-1β receptor coupled to a reporter gene in A549 cells (Buell et al., 1998). Data are the mean±s.e.m of 3 experiments.

The potency of dog IL-1β (pEC50 = 9.3±0.1) at the human IL-1β receptor of A549 cells was 10 fold lower than human IL-1β (pEC50 = 10.2±0.1, t-test, p<0.05) but the response could be used to bioassay dog IL-1β in dog blood. ATP-stimulated IL-1β release in dog and human whole blood was relatively rapid in onset (measurable within 10-20 min of agonist addition) and reached a plateau after 20 min in human and 40 min in dog whole blood. There was no clear dependence of EC50 on time of incubation with ATP between 20 and 60 min (One way ANOVA, p>0.05). Following a 20 min incubation the pEC50 for ATP in dog whole blood (3.09±0.07) was similar to that in human whole blood (pEC50 = 2.9±0.04, t-test, p>0.05). KN62 is a relatively potent antagonist of both human and dog P2X7 receptors (Roman et al., this meeting) and it also non-competitively blocked responses to ATP in human and dog whole blood (pIC50 values were 6.7±0.1 and 5.75±0.1 for inhibiting responses to 2mM ATP respectively in dog and human blood).

In this study we have demonstrated that high concentrations of ATP stimulate IL-1β release in LPS-primed human and dog whole blood. The low potency of ATP is suggestive of activation of a P2X7 receptor though this interpretation is confounded by potential metabolism of ATP. It proved possible to evaluate P2X7 antagonist effects in whole blood and the relatively high potency of KN62 provides further evidence that that the response to ATP in whole blood is mediated via the P2X7 receptor.

Buell, G. et al, (1998). Blood, 92, 3521-3528.

Labasi, J.M. et al, (2002). J Immunol, 168, 6436-6445.



Conflict of interest: The authors are employed by GlaxoSmithKline R&D Ltd.