Regulation Of Thymic Stromal Cells On Heat Stress-treated Thymocytes In Vitro

Objective To explore the effects of thymic stromal cells (TSC) on normal or heat stress-treated thymocytes and their mechanism in vitro. Methods Thymocytes from 5-week-old BALB/c male mice were incubated at 430C for 1 hour, then cocultured with TSC or supernatant of TSC (TSC-SN) at 37 0C. Thymocytes were recollected and counted after cocultured with TSC or TSC-SN at the 0, 6th, 12th , 24th or 48th h. The apoptotic rate, CD4CD8 subpopulations and the positive ratios of Fas, Fas-L and HSP7O expression thymocytes were assayed by flow cytometry (FCM). The interactions between TSC and thymocytes were also observed by light and electron microscopy. Results (1) The apoptotic rate and the positive ratios of Fas, Fas-L and HSP7O expression increased in heat stress-treated thymocytes in vitro. The expression of Fas, Fas-L on thymocytes is significantly associated with the apoptosis of thymocytes. (2) After cocultured with TSC, the viability of thymocytes increased, while the ratios of apoptosis and the positive ratios of Fas, Fas-L expression thymocytes decreased in non-heat-stress-treated thymocytes. At the same time, the counts of thymocytes and the immature CD4~CD8~ double positive (DP) thymocytes abruptly decreased, although the ratios of CD4~CD8 and CDtCD8~ single positive (SP) thymocytes were higher than before. (3) When cocultured with TSC, the positive ratio of HSP7O expression in heat stress-treated thymocytes increased obviously, while both DP and SP thymocytes decreased significantly. The ratio of apoptosis, the positive ratios of Fas and Fas-L expression in this group also decreased, but they were much higher than those in non-heat-stress-treated thymocytes. (4) Whether thymocytes were treated with heat stress or not, the adhesion of thymocytes to TSC and engulfing thymocytes by TSC were observed by light and electron microscope. (5) When cultured in 50% TSC- SN, the Fas expression thymocytes and the ratios of apoptosis decreased in -5- heat stress-treated or non-heat-stress-treated thymocytes, while the expression of Fas-L lowered on heat stress-treated thymocytes. No obvious change of the expression of HSP7O was observed in the two groups. Conclusion (1) Fas/Fas-L mediated apoptosis is an important pathway of spontaneous or heat stress induced death of thymocytes. Immature DP thymocytes are the major apoptotic cells induced by heat stress. HSP7O is a marker of damaged cells in heat stress treated thymocytes. (2) Through direct contacting, TSC play an important role on elimination of DP thymocytes by engulfing and differentiation of some DP thymocytes into SP thymocytes. (3) Through direct contacting, TSC eliminate both heat stress- treated DP and SP thymocytes. The increasing expression of HSP7O on heat stress-treated thymocytes cocultured with TSC may exert dual functions. One is protection of thymocytes from apoptosis, the other is recognition and engulfing demaged or apoptotic thymocytes. It is possible that HSP7O plays an important role in antigen presentation during the damaged or apoptotic SP thymocytes were engulfed by TSC. (4) The protection of TSC against the apoptosis of thymocytes was stronger in non-heat-stress-treated group than heat stress treated group. The close relationship between Fas/Fas-L expression and apoptosis of thymocytes suggests an important mechanism of clone deletion of thymocytes. (5) TSC-SN also showed some effects on preven...