FLOW CYTOMETRIC ANALYSIS OF CD3 TCR COMPLEX, ZINC, AND GLUCOCORTICOID-MEDIATED REGULATION OF APOPTOSIS AND CELL-CYCLE DISTRIBUTION IN THYMOCYTES FROM OLD MICE/

Apoptosis represents the main mechanism involved in the intrathymic ce ll selection. The involution and atrophy of the thymic gland during ag ing has been associated with an altered representation of thymocyte su bsets and particularly of CD4(+) CD8(+) double-positive (DP) thymocyte s, i.e., the cell population mainly involved in thymocyte selection. T he aim of the present study was to evaluate the responsiveness of thym ocytes from old mice to factors regulating the apoptotic cell death, s uch as antibodies to CD3/T cell receptor complex, zinc, and dexamethas one (DEX), Balb/c mice were used at the ages of 2 months (young), 21-2 2 months (old), and 24-26 months (very old), Thymocytes from these mic e were: incubated overnight with anti-CD3 monoclonal antibody (8 mu g/ ml), Zn2+ (150 mu M), or DEX (10(-7) M) and then analysed for number o f apoptotic nuclei, cell cycle phase, and phenotype by flow cytometry. A significant decrease of both the total number and the proportion of DP thymocytes was present in old and very old mice in comparison with young animals. Antibodies to CD3 antigen induced thymocyte apoptosis in both young and old mice. The stimulation of the CD3/TCR complex was more effective in giving apoptosis in very-old than in old and young mice. An impairment of the inhibiting effect of zinc on apoptosis indu ced by either serum deprivation or DEX was found in old and very old m ice, whereas zinc was less effective in inhibiting CD3-induced apoptos is only in very Old animals. Reduced DES-induced apoptosis was also pr esent in old age; this effect was more evident in very old than in old mice. Thymocyte apoptosis in old mice required protein synthesis bein g blocked With cycloheximide, Apoptosis was exerted on thymocytes in a specific cell cycle phase, i.e., on G0/G1 phase cells. Anti-CDS antib odies, Zn2+, or DEX regulated apoptosis by modulating the proportion o f DP thymocytes, The results demonstrate an altered in vitro responsiv eness of thymocytes from old and very old mice to factors regulating a poptosis and suggest further investigations to determine if this alter ed responsiveness is associated with increased apoptosis of thymocyte populations occurring with increasing age. (C) 1998 Wiley-Liss, Inc.