Detection of in situ mitotic activity of dendritic epidermal T-cells by BrdU labeling

We analyzed mitotic dendritic epidermal T-cells (DETC) in the epidermis of C3H/He (Thy-1.2+) mice, using double immunoenzymatic labeling. Ear skin was incubated with 100 microM bromodeoxyuridine (BrdU) for 5 hr and then either directly studied or cultured for an additional 12 hr in BrdU-free medium. After BrdU labeling, with or without additional culture, epidermal sheets were obtained by ethylenediaminetetraacetic acid separation. The epidermal specimens were immunostained by the peroxidase method to visualize nuclear BrdU and then by the biotin-streptavidin-alkaline phosphatase method for surface Thy-1.2 antigen. In specimens processed immediately after BrdU labeling, a mean 3.0% of all basal cells were labeled with BrdU and a mean 1.1% of the BrdU-labeled cells were also positive for Thy-1.2. Moreover, a mean 2.1% of the DETC had incorporated BrdU. BrdU-labeled DETC had a variety of appearances; they were dendritic and round in the BrdU-treated specimens, while oval and paired cells were also found in the specimens after additional culture. These morphological changes of BrdU-labeled DETC demonstrate that resident DETC can become mother cells undergoing mitosis through the retraction of their dendrites, and it appears that DETC divide at a relatively high rate, i.e., up to 10% of the DETC may enter the S-phase of the cell cycle every 24 hr.