Human P2Y11 Expression Level Affects Human P2X7 Receptor-Mediated Cell Death

Abstract

Adenosine triphosphate (ATP) is known to induce cell death in T lymphocytes at high extracellular concentrations. CD4⁺ and CD8⁺ T lymphocytes have a differential response to ATP, which in mice is due to differences in the P2X7 receptor expression levels. By contrast, we observed that the difference in human CD4⁺ and CD8⁺ T lymphocyte response toward the synthetic ATP-analog BzATP is not explained by a difference in human P2X7 receptor expression. Rather, the BzATP-induced human P2X7 receptor response in naïve and immune-activated lymphocyte subtypes correlated with the expression of another ATP-binding receptor: the human P2Y₁₁ receptor. In a recombinant expression system, the coexpression of the human P2Y₁₁ receptor counteracted BzATP-induced human P2X7 receptor-driven lactate dehydrogenase release (a marker of cell death) and pore formation independent of calcium signaling. A mutated non-signaling human P2Y₁₁ receptor had a similar human P2X7 receptor-inhibitory effect on pore formation, thus demonstrating that the human P2X7 receptor interference was not caused by human P2Y₁₁ receptor signaling. In conclusion, we demonstrate an important species difference in the ATP-mediated cell death between mice and human cells and show that in human T lymphocytes, the expression of the human P2Y₁₁ receptor correlates with human P2X7 receptor-driven cell death following BzATP stimulation.

Keywords: A740003; Fluo-4; P2RX7; P2RY11; YO-PRO-1; cyclic adenosine monophosphate; purinergic.

Figure 1

CD4⁺ and CD8⁺ T lymphocytes show a differential response to P2X7 stimulation. (A) The difference in CD4⁺ (orange rhomb) and CD8⁺ (purple square) T lymphocyte P2X7 receptor activation with BzATP was measured as lactate dehydrogenase (LDH) activity in the supernatant following 2 h stimulation. Data are shown as mean ± SEM normalized to maximum induced cell death in 1% Triton X-100, n = 5–6 from three independent donors. (B) The BzATP response in CD4⁺ and CD8⁺ T lymphocytes blocked by P2X7 receptor antagonist, A740003. Cells were stimulated with 1,000 µM BzATP in combination with various concentrations of P2X7 receptor antagonist, A740003, and P2X7 receptor activation was measured as the activity of LDH after 2 h. Data are shown as mean ± SEM of maximum induced cell death at 1% Triton X-100, n = 11–14 from seven independent donors. LDH baseline activity is shown as solid lines for CD4⁺ (orange) and CD8⁺ (purple) T lymphocytes, respectively.

Figure 2

P2X7 levels are equally high in CD4⁺ and CD8⁺ T lymphocytes. (A) P2RX7 gene expression in human CD4⁺ and CD8⁺ T lymphocytes shown as ΔCt values normalized to the genes for GAPDH and ACTB (β-actin) from 18 to 21 independent donors. (B) Western blot showing P2X7 receptor signal (75 kDa) from protein lysate of CD4⁺ (orange) and CD8⁺ (purple) T lymphocytes from five different donors (#1–5). Loading control was β-actin (42 kDa).

Figure 3

Higher P2Y₁₁ levels in CD8⁺ T lymphocytes and immune-activated CD4⁺ T lymphocytes correlate with lower responses to BzATP compared to naïve CD4⁺ T lymphocytes. (A) Left axis: P2RY11 gene expression in human CD4⁺ and CD8⁺ T lymphocytes shown as ΔCt values normalized to the genes for GAPDH and ACTB (β-actin) from 14 to 18 independent donors. Right axis: mean fluorescence intensity (MFI) as measured by flow cytometry on eight independent donors. (B) Western blot membrane with protein lysate of CD4⁺ (orange) and CD8⁺ (purple) T lymphocytes from five different donors (#1–5) following stripping and re-blotting for P2Y₁₁ signal (33 kDa). Loading control was β-actin (42 kDa) after stripping and re-blotting. (C) ΔCt values for human P2RY11 (green) and P2RX7 (red) gene expression over time in naïve and immune-activated CD4⁺ T lymphocytes. Values were normalized to the genes for B2M and EEF1A1 from three independent donors. (D) Lactate dehydrogenase (LDH) activity in the supernatant following 2 h incubation with various concentrations of BzATP of 2-day cultured naïve (filled rhomb) or immune-activated (open rhomb) CD4⁺ T lymphocytes. Data are shown as mean ± SEM of maximum induced cell death at 1% Triton X-100, n = 4–6 from three independent donors.

Figure 4

P2RY11 silencing in CD8⁺ T lymphocytes shows increased BzATP-induced cell death following immune activation. (A) Relative P2RY11 gene expression changes 24 and 48 h in CD8⁺ T lymphocytes left untreated (gray) or after transient transfection with non-targeting (NT) (turquoise) or siRNA targeting P2RY11 (purple). (B) P2Y₁₁ protein expression measured by flow cytometry count in isotype control (black), NT siRNA (turquoise), NT siRNA immune-activated (red), P2RY11-silenced (purple), or P2RY11-silenced immune-activated CD8⁺ T lymphocytes from a single donor. (C) Mean fluorescence intensity (MFI) from P2Y₁₁ receptor protein expression measured by flow cytometry on transient transfected CD8⁺ T lymphocytes. (D) Lactate dehydrogenase (LDH) activity in transiently transfected CD8⁺ T lymphocytes stimulated 2 h with BzATP. Data are shown as mean ± SEM of maximum induced cell death at 1% Triton X-100 following baseline correction, n = 5–6 from two independent donors.

Figure 5

P2X7-mediated cell death and pore formation are lower when cells are cotransfected with P2RY11. Top row: the activity of lactate dehydrogenase (LDH) in the supernatant after 2 h in human embryonic kidney-293 (HEK-293) cells transiently transfected with vectors expressing purinergic receptors. Cells were stimulated with (A) increasing doses of BzATP, n = 5–6 from three experimental runs or (B) 1,000 µM BzATP in combination with various concentrations of P2X7 receptor antagonist, A740003, n = 6–8 from five experimental runs. Data are shown as mean ± SEM of maximum-induced cell death by 1% Triton X-100. LDH baseline activity is shown as solid lines for eGFP + empty vector (black), P2RX7 + empty vector (red), and P2RX7 + P2RY11 (blue), respectively. Bottom row: GripTite cells transiently transfected with vectors expressing purinergic receptors were stimulated with various concentrations of BzATP to induce pore formation measured as YO-PRO-1 fluorescent dye uptake. Cells were stimulated with (C) increasing doses of BzATP, n = 12–29 from nine experimental runs or (D) BzATP and P2X7 receptor antagonist, A740003, n = 6–15 from five experimental runs. Data are shown as mean ± SEM.

Figure 6

P2X7-mediated Ca²⁺ response is still present when cells are cotransfected with P2RY11. Normalized calcium response measured by Fluo-4 following stimulation with various concentrations of BzATP of transiently transfected GripTite cells. Data are shown as mean ± SD, n = 9 from three independent experiments. Dose–response curves for P2RX7 (red) and P2RY11 (green) were fitted to a sigmoidal curve and cotransfected P2RX7 + P2RY11 to a biphasic sigmoidal curve. Vertical dashed lines represent EC₅₀ values for respective curves and 95% confidence intervals in colored shades. See text for details.

Figure 7

The effect of P2Y₁₁ on P2X7-mediated pore formation is not dependent on P2Y₁₁ signaling. Stimulation with various concentrations of BzATP of transiently transfected GripTite cells expressing empty vector (black), P2RY11 (closed green square), or P2RY11 C919T (open green square) vector in (A) calcium assay measured by Fluo-4, data are shown as mean ± SEM, n = 3,6 from 1 to 2 independent runs. (B) cAMP response, data are shown as mean ± SEM, n = 3,6 from 1 to 2 independent runs. (C) YO-PRO-1 dye uptake as a measure of P2X7 receptor pore formation in cells transfected with P2RX7 + empty vector (red), P2RX7 + P2RY11 (half-open blue triangle), and P2RX7 + P2RY11 C919T (open blue triangle). Data are shown as mean ± SEM, n = 9 from three independent runs. Part of data was previously published by Ref. (25).