Ticagrelor potentiates adenosine-induced stimulation of neutrophil chemotaxis and phagocytosis

Abstract

In the PLATO study, ticagrelor was associated with fewer pulmonary infections and subsequent deaths than clopidogrel. Neutrophils are a first-line defence against bacterial lung infection; ticagrelor inhibits cellular uptake of adenosine, a known regulator of neutrophil chemotaxis and phagocytosis. We assessed whether the inhibition of adenosine uptake by ticagrelor influences neutrophil chemotaxis and phagocytosis. Neutrophils and erythrocytes were isolated from healthy volunteers. Concentration-dependent effects of adenosine on IL-8-induced neutrophil chemotaxis were investigated and the involved receptors identified using adenosine receptor antagonists. The modulatory effects of ticagrelor on adenosine-mediated changes in neutrophil chemotaxis and phagocytosis of Streptococcus pneumoniae were determined in the presence of erythrocytes to replicate physiological conditions of cellular adenosine uptake. Low-concentration adenosine (10(-8)M) significantly increased IL-8-induced neutrophil chemotaxis (% neutrophil chemotaxis: adenosine 28.7%±4.4 vs. control 22.6%±2.4; p<0.01) by acting on the high-affinity A1 receptor. Erythrocytes attenuated the effect of adenosine, although this was preserved by ticagrelor and dipyridamole (another inhibitor of adenosine uptake) but not by control or by cangrelor. Similarly, in the presence of erythrocytes, a low concentration of adenosine (10(-8)M) significantly increased neutrophil phagocytic index compared to control when ticagrelor was present (37.6±6.6 vs. 28.0±6.6; p=0.028) but had no effect in the absence of ticagrelor. We therefore conclude that the inhibition of cellular adenosine reuptake by ticagrelor potentiates the effects of a nanomolar concentration of adenosine on neutrophil chemotaxis and phagocytosis. This represents a potential mechanism by which ticagrelor could influence host defence against bacterial lung infection.

Keywords: Adenosine; Dipyridamole; Erythrocytes; Neutrophils; Ticagrelor.

Graphical abstract

Fig. 1

Effects of IL-8 and adenosine on neutrophil chemotaxis. Chemotactic response of neutrophils to increasing concentrations of IL-8 (A; n = 4) or adenosine (B; n = 4). The effect of increasing concentrations of adenosine on neutrophil chemotaxis induced by IL-8 10^− 9 M (C; n = 8). The number of neutrophils that migrated over 30 min was counted and results expressed as a percentage of the total number of neutrophils added to the filter membranes of chemotaxis chambers. Results are presented as mean ± SEM and analysed for statistical significance using one-way analysis of variance followed by Dunnett's t-test. **p < 0.01 and ***p < 0.001 compared to control.

Fig. 2

Effect of adenosine receptor antagonists on neutrophil chemotaxis in the presence of adenosine. The effect of the A₁ antagonist DPCPX 10^− 7 M (A and B), the A₂ antagonist SCH58261 10^− 7 M (C and D) and the A₃ antagonist MRS 1334 10^− 7 M (E and F) on neutrophil migration to IL-8 10^− 9 M over 30 min in the presence or absence of adenosine 10^− 8 M (A, C and E; n = 6) or adenosine 10^− 5 M (B, D and F; n = 7). All inhibitors were assessed within the same experiment but are divided into different panels for clarity. Results are presented as mean ± SEM and analysed for statistical significance using one-way analysis of variance followed by Bonferroni's test for multiple comparisons. *p < 0.05 and **p < 0.01.

Fig. 3

Effect of erythrocytes on the response to adenosine. Chemotactic response of neutrophils to IL-8 10^− 9 M, in the presence of adenosine 10^− 8 M and in the absence (white columns) or presence (black columns) of erythrocytes (n = 14). Results are presented as mean ± SEM and analysed for statistical significance using one-way analysis of variance followed by Bonferroni's test for multiple comparisons. **p < 0.01 and *** < 0.001.

Fig. 4

Effects of cangrelor, ticagrelor and dipyridamole on neutrophil chemotaxis in the presence of erythrocytes and the absence or the presence of adenosine. Chemotactic response of neutrophils to IL-8 10^− 9 M, in the presence of erythrocytes and without (white columns) or with (black columns) addition of adenosine 10^− 8 M, showing the effects of cangrelor, ticagrelor and dipyridamole all at concentrations of either (A) 10^− 5 M or (B) 10^− 6 M, compared to vehicle control (n = 7). Results are presented as mean ± SEM and analysed for statistical significance using one-way analysis of variance followed by Bonferroni's test for multiple comparisons.*p < 0.05, ***p < 0.001 and **** < 0.0001.

Fig. 5

Effect of ticagrelor on changes in neutrophil phagocytosis induced by low and high concentrations of adenosine in the presence of erythrocytes. Effect of ticagrelor (10^− 5 M) on changes in neutrophil phagocytosis of S. pneumoniae, determined by percentage of neutrophils containing phagocytosed S. pneumoniae (A) and phagocytic index (B), induced by 10^− 8 M and 10^− 5 M adenosine in the presence of erythrocytes (n = 8). Results are expressed as mean ± SEM and analysed for statistical significance using two-way ANOVA followed by Bonferroni's test for multiple comparisons. *p < 0.05, **p < 0.01.

Fig. 6

Interaction between ticagrelor and the A1 receptor antagonist DPCPX. Effect of ticagrelor (10^− 5 M) and DPCPX (10^− 7 M) on neutrophil phagocytosis of S. pneumoniae determined by percentage of neutrophils containing phagocytosed S. pneumoniae (A) and phagocytic index (B), induced by 10^− 8 M adenosine in the presence of erythrocytes (n = 5). Results are expressed as mean ± SEM and analysed for statistical significance using two-way ANOVA followed by Bonferroni's test for multiple comparisons. *p < 0.05, **p < 0.01.