Presence of B220 within thymocytes and its expression on the cell surface during apoptosis

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Abstract
B220 is the full-length splicing isoform of a tyrosine phosphatase CD45 and is predominantly expressed as a transmembrane protein on B cells. Other splicing isoforms of CD45 are yielded by alternative splicing of exons 4, 5 and 6. Recently, the expression of B220 on peripheral T cells during activation-induced cell death has been reported. To investigate whether B220 is implicated in apoptosis of immature T cells, we analysed (by flow cytometry using the anti-B220 monoclonal antibody, RA3-6B2) the expression of B220 on mouse thymocytes undergoing X-irradiation- and dexamethasone (DEX)-induced apoptosis. The expression of B220 on thymocytes positive for Thy-1 was induced by X-irradiation or DEX treatment and increased with length of incubation. The expression of B220 was pronounced on the apoptotic hypodiploid cells in the fraction showing lower forward scattering values. Reverse transcription–polymerase chain reaction detected mRNA containing exons 4, 5 and 6 of CD45 in normal thymocytes as well as those exposed to X-rays or DEX. Surprisingly, cytoplasmic B220 antigens were detected in a considerable fraction of normal thymocytes. Moreover, the expression level of the 220 000-MW protein in normal thymocytes was similar to that in the thymocytes undergoing apoptosis. During apoptosis, the expression level of B220 antigen was reduced in the cytoplasm but, conversely, up-regulated on the surface of thymocytes. These results suggest that B220 is constitutively expressed as a cytoplasmic form within thymocytes and possibly translocated to the cell membrane during apoptosis.