Novel method for real-time monitoring of ATP release reveals multiple phases of autocrine purinergic signalling during immune cell activation.

AIMS: The activation of immune cells must be tightly regulated to allow an effective immune defence while limiting collateral damage to host tissues. Cellular ATP release and autocrine stimulation of purinergic receptors are recognized as critical regulators of immune cell activation. However, the study of purinergic signalling has been hampered by the short half-life of the released ATP and its breakdown products as well as the lack of real-time imaging methods to study spatiotemporal dynamics of ATP release. METHODS: To overcome these limitations, we optimized imaging methods that allow monitoring of ATP release with conventional microscopy using the recently developed small molecular ATP probes 1-2Zn(II) and 2-2Zn(II) for imaging of ATP in the extracellular space and release at the surface of living cells. RESULTS: 1-2Zn(II) allowed imaging of <1 µm ATP in the extracellular space, while 2-2Zn(II) provided unprecedented insights into the spatiotemporal dynamics of ATP release from neutrophils and T cells. Stimulation of these cells caused virtually instantaneous ATP release, which was followed by a second phase of ATP release that was localized to the immune synapse of T cells and the leading edge of polarized neutrophils. Imaging these ATP signalling processes along with mitochondrial probes provided evidence for a close spatial relationship between mitochondrial activation and localized ATP release in T cells and neutrophils. CONCLUSION: We believe that these novel live cell imaging methods can be used to define the roles of purinergic signalling in immune cell activation and in the regulation of other complex physiological processes.