Development of intravital intermittent confocal imaging system for studying Langerhans cell turnover

Although several studies have suggested relatively slow turnover of Langerhans cells (LCs), their actual lifespan remains elusive. Here we report the development of a new intravital imaging system for studying LC efflux and influx. Epidermal LCs expressing enhanced green fluorescent protein (EGFP) were visualized in anesthetized I-A beta-EGFP knock-in mice by confocal microscopy. By overlaying two sets of EGFP(+) LC images recorded in the same microscopic fields at time 0 and 24 hours later, we identified LC subpopulations that had disappeared from or newly emerged in the epidermis during that period. Of > 10,000 LCs analyzed in this manner, an overwhelming majority (97.8 +/- 0.2%) of LCs showed no significant changes in the x-y locations, whereas 1.3 +/- 0.1% of the LCs that were found at time 0 became undetectable 24 hours later, representing LC efflux. Conversely, 0.9 +/- 0.1% of the LCs that were found at time 24 hours were not detectable at time 0, representing LC influx. From these frequencies, we estimated the half-life of epidermal LCs to range from 53 to 78 days, providing new insights into the immunobiology of LCs. Our intermittent imaging approach may be regarded as a technical breakthrough enabling direct visual assessment of LC turnover in living animals.