Recruitment of macrophages towards sites of inflammation by chemoattractant signals is critical for an effective innate immune response. The molecular mechanism by which macrophages navigate chemotactic gradients is not well understood. Recently, Chen et al. (2006) reported that autocrine ATP release and P2Y₂ receptor stimulation play important roles in neutrophil chemotactic orientation. Extracellularly released ATP can activate multiple purinergic receptors since it is sequentially degraded to ADP, AMP and adenosine by ecto-nucleotidases. Here, we investigated whether autocrine ATP signaling was part of the macrophage navigation system using a novel chemotaxis chamber from Ibidi (Martinsried, Germany). Using Alexa Fluor 488-conjugated 10,000 MW dextran and confocal microscopy we found that gradients were steep and stable within 6 h. Resident peritoneal macrophages were seeded into the narrow channel (and observation area) separating the two 40 ml reservoirs of the chemotaxis chamber, and the chemoattractant C5a was introduced into one of the two reservoirs. Macrophages were imaged by phase-contrast microscopy using a Zeiss AxioObserver microscope fitted with a temperature-controlled stage incubator, and migration tracks between 6 and 12 h were analyzed using ImageJ (NIH). Chemotaxis was abolished by the ATPase/ADPase apyrase, implying that ATP release and autocrine purinergic signaling is indeed essential for chemotactic sensing. Moreover, we could reproduce the apyrase effect by blocking multiple purinergic feedback loops. Thus, we show that the autocrine ATP-ADP-adenosine signaling axis is critical for macrophage chemotactic navigation.

Y. Chen et al. Science 314, 1792–1795 (2006).