Autocrine regulation of T-cell activation by ATP release and P2X7 receptors

Abstract

T-cell activation requires the influx of extracellular calcium, although mechanistic details regarding such activation are not fully defined. Here, we show that P2X(7) receptors play a key role in calcium influx and downstream signaling events associated with the activation of T cells. By real-time PCR and immunohistochemistry, we find that Jurkat T cells and human CD4(+) T cells express abundant P2X(7) receptors. We show, using a novel fluorescent microscopy technique, that T-cell receptor (TCR) stimulation triggers the rapid release of ATP (<100 microM). This release of ATP is required for TCR-mediated calcium influx, NFAT activation, and interleukin-2 (IL-2) production. TCR activation up-regulates P2X(7) receptor gene expression. Removal of extracellular ATP by apyrase or alkaline phosphatase treatment, inhibition of ATP release with the maxi-anion channel blocker gadolinium chloride, or siRNA silencing of P2X(7) receptors blocks calcium entry and inhibits T-cell activation. Moreover, lymphocyte activation is impaired in C57BL/6 mice that express poorly functional P2X(7) receptors, compared to control BALB/c mice, which express fully functional P2X(7) receptors. We conclude that ATP release and autocrine, positive feedback through P2X(7) receptors is required for the effective activation of T cells.

Figure 1.

Stimulation of Jurkat cells releases cellular ATP. A) Jurkat cells were stimulated by ligation of CD3/CD28 using MACSibeads coated with anti-CD3 and anti-CD28 antibodies (arrowheads), and extracellular ATP levels near the cell surface were visualized with a fluorescence microscope assay and estimated using ATP standards. B) Extracellular ATP concentrations determined from the indicated areas of interest shown in A. C) Changes in ATP, ADP, AMP, and adenosine concentrations as a function of time in the bulk cell supernatant of Jurkat cells (5×10⁶) treated with anti-CD3 and anti-CD28 antibodies (1 μg/ml each) were measured with HPLC. Data shown in A and B are representative of 3 and 2 similar experiments, respectively, performed in separate experiments.

Figure 2.

Extracellular ATP is required for T-cell activation. A) IL-2 mRNA levels of Jurkat cells (10⁷) stimulated for 1 h with anti-CD3/CD28 antibody-coated beads (1 bead/cell) in the presence or absence of 100 μM ATP. B) Inhibition of ATP release from Jurkat cells (10⁷) treated with the maxi-anion channel blocker, GdCl₃ (100 μM). Cells were treated in HBSS for 20 min at 37°C and stimulated with anti-CD3/CD28 antibodies (1 μg/ml each) for 1 min, and ATP release was determined using the ATP Bioluminescence Assay Kit HSII. C) GdCl₃ (100 μM) and apyrase (20 U/ml) inhibit IL-2 mRNA expression of stimulated Jurkat cells and IL-2 production of stimulated human PBMCs. IL-2 mRNA expression was determined by real-time RT-PCR analysis, and IL-2 production of PBMCs was determined with ELISA. Data are means ± se. *P ≤ 0.05; n = 3; two-tailed unpaired Student’s t test.

Figure 3.

Scavenging of released ATP inhibits calcium signaling and IL-2 expression. A, B) Scavenging extracellular ATP with 10 U/ml of apyrase (A) or alkaline phosphatase (ALP; B) inhibits intracellular Ca²⁺ signaling of Jurkat cells stimulated with anti-CD3 antibodies. C) Apyrase and ALP suppressed IL-2 mRNA transcription in response to stimulation of Jurkat cells by ligation of CD3 and CD28 for 4 h. IL-2 mRNA levels were determined by real-time RT-PCR, and intracellular Ca²⁺ mobilization was measured using Fura-2. Data shown in A and B are representative of 3 similar experiments performed separately. Data shown in C represent means ± se. *P ≤ 0.05; n = 3; two-tailed unpaired Student’s t test.

Figure 4.

P2X receptor antagonists inhibit Ca²⁺ signaling and IL-2 production. A, B) The nonselective P2X receptor antagonists suramin (100 μM) and NF023 (10 μM) and the P2X₇-selective antagonist o-ATP (10 μM) blocked Ca²⁺ signaling in Jurkat cells stimulated with 2 mM ATP (A) or 1 μg/ml anti-CD3 antibodies (B). C) Suramin and o-ATP inhibited IL-2 production of stimulated PBMCs. Cells were treated with antagonists for 1 h prior to cell stimulation. Ca²⁺ mobilization was measured using Fura-2, and IL-2 production of PBMCs was determined by ELISA. Data in A and B are representative of 3 separate experiments; data in C represent means ± se. *P ≤ 0.05; n = 3; two-tailed unpaired Student’s t test.

Figure 5.

P2X₇ receptors are involved in T-cell activation. A) P2X₇ receptor mRNA expression in Jurkat cells and human peripheral CD4⁺ T cells was quantified using real-time RT-PCR analysis. B) Immuncytochemical assessment of P2X₇ receptor expression of Jurkat cells and human CD4⁺ T cells was evaluated using a fluorescence microscope. Controls were probed with goat anti-rabbit Alexa 555 secondary antibody only. C) P2X₇ receptor mRNA expression of Jurkat cells was determined with real-time RT-PCR 4 h after stimulation with or without anti-CD3/CD28 antibody-coated beads. D) NFAT activation of Jurkat cells overexpressing wild-type (WT) or mutated (T1729A) P2X₇ receptor or empty expression vector (control) was assessed using a NFAT-luciferase reporter assay. Cells were stimulated with anti-CD3/CD28 antibody-coated beads. NFAT-luciferase activity was normalized using a β-galactosidase control reporter coexpressed in the cells. Transfected cells were cultured for 72 h, stimulated with anti-CD3/CD28 antibody-coated beads for 8 h, and luciferase activity was measured. Data represent means ± se of triplicates. *P ≤ 0.05; n ≥ 3; two-tailed unpaired Student’s t test.

Figure 6.

P2X₇ receptors are involved in Ca²⁺ signaling and IL-2 expression. A) Jurkat cells electroporated with a Cy3-labeled nonsense siRNA construct were examined using a microscope to verify siRNA uptake. B) Jurkat cells were transfected with 3 μg siRNA targeting P2X₇ or nonsense siRNA, and P2X₇ gene expression was examined at the indicated time points by assessing mRNA levels using real-time RT-PCR. C) Jurkat cells were treated with nonsense control siRNA or with siRNA targeting P2X₇ receptors for 72 h, then stimulated with anti-CD3/CD28 antibody-loaded beads, and NFAT-luciferase activation,and IL-2 transcription were determined. Data represent means ± se. *P ≤ 0.05; n = 3; two-tailed unpaired Student’s t test. D, E) Jurkat cells were treated with siRNA to silence P2X₇ receptors or with nonsense control siRNA, and Ca²⁺ signaling in response to stimulation with 0.5 μg/ml anti-CD3 antibody (D) or 2 mM ATP (E) was determined by flow cytometry using Fluo-3 as a Ca²⁺ indicator. Data are representative of similar results obtained in 3 separate experiments.

Figure 7.

P2X₇ receptors regulate IL-2 production in mouse splenocytes and a model for the role of ATP release and P2X₇ receptors in T-cell activation. A) IL-2 expression of splenocytes from C57BL/6 mice lacking functional P2X₇ receptors or BALB/c mice with functional P2X₇ receptors was assessed using intracellular cytokine staining and flow cytometry. Splenocytes were stimulated overnight with the indicated concentrations of phytohemagglutinin (PHA), and IL-2 expression of lymphocytes was assessed by intracellular staining. Data are representative of 3 experiments; values are means ± se of triplicate measurements. B) Schematic depiction of the role of ATP release and P2X₇ receptors in T-cell activation. TCR activation by antigen presenting cells (APC) induces ATP release, resulting in the activation of P2X₇ receptors that facilitate Ca²⁺ entry, which, in turn, induces T-cell activation events, through the activation of NFAT and IL-2 transcription.