Removal of inflammatory neutrophils from sites of inflammation can occur by a number of routes; into exudates, by apoptosis followed by macrophage clearance and by retrograde chemotaxis. The relative contribution of these disposal mechanisms in vivo has been hard to define, and the lifespan of an in vivo tissue neutrophil has been hard to directly measure. We have generated transgenic zebrafish expressing the fluorescent photo-convertible protein, Kaede, in neutrophils.
Objective To label individual inflammatory neutrophils and track their fate during inflammation resolution in vivo.
Method Individual neutrophils were marked by photoconverting the Kaede protein using 405 nm laser light restricted to the individual cell profiles. Known numbers of neutrophils were photoconverted and visualised over 48 h. In subsequent experiments, an inflammatory reaction was induced by sterile tail transection of transparent zebrafish larvae. Kaede labelled neutrophils are recruited to the site of injury where they can be photoconverted and followed using time lapse video microscopy.
Results By counting the number of remaining photoconverted neutrophils over time, the half-life of a neutrophil was calculated. Our data suggest the lifespan of a zebrafish neutrophil in the tissues is 117.7 (CI 95.67 to 157.8) h, a figure comparable to that inferred for human tissue neutrophils. Timelapse videos reveal a population of neutrophils that migrate away from the site of injury, undergoing retrograde chemotaxis. Whilst neutrophils can migrate away from the site of injury, they are not completely free to do so. The apparent restriction on their behaviour may be due to the presence of a persisting chemical gradient or may reflect an intrinsic feature of neutrophil behaviour.
Conclusions These data demonstrate the power of this model to inform our understanding of phagocyte behaviour and interaction in vivo.
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