Regulatory Effect of CD9 on Calcium-Stimulated Phosphatidylserine Exposure in Jurkat T Lymphocytes

Abstract

Alteration in membrane-phosphatidylserine (PS) asymmetry occurs during lymphocyte apoptosis, but the mechanism and regulation of this process are not well understood. We investigated the possible involvement of CD9, a member of the tetraspan family of membrane proteins, in the PS-exposure process in Jurkat T cells; flow cytometry with FITC–annexin V was used to detect the PS-positive cells. We found that antibody to CD9 inhibited the prompt (within 5 min) PS exposure stimulated by calcium ionophore A23187, whereas it had no effect on resting cells. Antibodies against other control antigens (CD7, CD56, CD57, CD59, CD63, and CD71) had no effect on PS exposure in either resting or calcium-ionophore-treated cells. The inhibitory effect of anti-CD9 was dose dependent. The observed inhibitory effect appeared to be “all or none” at the cellular level: increasing antibody doses decreased the percentage of PS-positive cells, whereas the number of PS molecules exposed per positive cell was constant. The inhibitory effect was not blocked by co-incubation with other antibodies of the same isotype, arguing against a nonspecific effect via Fc receptors. The anti-CD9, however, did not block the delayed (8- to 24-h) PS exposure induced by apoptotic treatments such as ultraviolet light, cycloheximide, and actinomycin D, indicating that CD9 might act selectively on only some pathways leading to PS exposure. Our results suggest that lymphocyte PS exposure is regulated by multiple pathways and that signals regulating PS exposure can be delivered through cell-surface antigen CD9.