Functional expression of P2X7 Receptor in T lymphocytes and its role in CD62L down-regulation

John Foster, Amanda Mackenzie, Stephen Ward. University of Bath, Bath, Somerset, UK.

The purinergic ATP-gated P2X7 receptor (P2X7) has previously been implicated in T lymphocyte functions. Studies with leukemic cell lines and mouse T lymphocytes has implicated P2X7 in lymphocyte activation, differentiation and cell death (Elliott et al., 2005; Schenk et al., 2008; Atarashi et al., 2008). Our aim was to investigate the pharmacology and function of P2X7 in primary human naive CD4+ T lymphocytes.

Electrophysiological responsiveness to concentrations of ATP known to activate P2X7 was assessed as described (Thompson et al., 2011). Naive CD4+ T lymphocytes were freshly isolated from peripheral human blood by negative selection using antibody/biotin microbead reagents from Miltenyi Biotec. In whole cell patch clamp recordings at the holding potential -60 mV, ATP (5mM, 10 s) application evoked an inward current with a current density of -2.32±2.90 pA/pF (n = 3). The absence of external CaCl2 and MgCl2 resulted in an increase in the ATP evoked current density to -61.96±98.88 pA/pF (n = 3). The P2X7 selective competitive antagonist, A438079 (10 μM), inhibited 5 mM ATP evoked currents (73.8±26.6 % inhibition, n = 3).

We next investigated functional responses to P2X7 activation by analysing shedding of CD62L (as assessed by measuring loss of its surface expression). Naive CD4+ cells (1x106/ml) were, pre-incubated with DMSO or inhibitors for 30 minutes and then stimulated with ATP. Cells were washed, labelled with anti-CD62L-FITC antibody and CD62L cell surface expression measured using flow cytometry. Mean fluorescent index was used as a measurement of surface CD62L expression and for the following experiments analysis was performed using one-way ANOVA and Tukey’s post test for significance between groups.

Application of 3 mM ATP resulted in a rapid (peak loss 15 minutes) decrease in cell surface CD62L expression (40.01±11.04 % loss after 15 minutes, n = 3) which was sustained for 1-3 hours. Non-cumulative dose response curves revealed a significant concentration-dependent down-regulation of CD62L in response to ATP (2-5 mM) treatment for 1 hour (p<0.001). Both A438079 and the non-competitive antagonist AZ11645373 inhibited CD62L down-regulation following 3 mM ATP (IC50 2.25±0.17μM and 1.35±0.277μM respectively, n = 3).

ADAM17 is the principle sheddase for CD62L. So, to explore its role in P2X7- mediated CD62L down-regulation, we used the broad spectrum matrix metalloproteinase inhibitor GM6001. At a concentration of 100μM, this compound significantly inhibited ATP induced CD62L down-regulation by 77.11±29.89 % (p<0.05 n = 3). No significant difference was observed when 0.1-5mM ATP was applied to cells with or without extracellular CaCl2 indicating that CD62L down-regulation was Ca2+ independent. Inhibitors of PKC, PI3K and MEK also had no effect on the ATP-induced loss of surface expressed CD62L.

Uncoupling mitochondrial complex I or III from the electron transport chain by 30 minute pre-incubation with Rotenone or Antimycin A had no significant effect on basal CD62L expression. However, but there was marked enhancement of ATP-induced loss of surface CD62L expression mediated by 5 μM Rotenone and 1μM Antimycin A (52.15 ± 5.78 % and 46.98 ± 10.74% increase) respectively, n = 3).

In conclusion human naive CD4 T lymphocytes express functional P2X7 that couples to surface CD62L down-regulation. We have discovered that altering the oxidative state of these cells effects ATP-mediated CD62L processing.

Elliott, J. I et al. (2005) Nat Cell Biol. (8):808-16

Schenk, U. et al. (2008) Sci Signal. 1(39):ra6

Atarashi, K. et al. (2008) Nature. 455(7214):808-12

Thompson et al. (2011) Cell Signal. Nov 18 [Epub ahead of print]

The authors would like to acknowledge the BBSRC and Pfizer for funding this project.